Antibiotic production (II)

ABSTRACT

The nucleic acid and amino acid sequences of α1, α2, β and ε subunits of acetyl-CoA carboxylase (ACCase) from  Streptomyces coelicolor  are provided. This subunit structure differs from that of known acyl carboxylases. Materials and methods are provided of increasing ACCase activity and production of secondary metabolites (such as polyketides and especially antibiotics) by causing expression in Streptomyces of such nucleic acid.  
     Also provided are methods of increasing ACCase activity and production of secondary metabolites (such as polyketides and especially antibiotics) by culturing Streptomyces in the presence of exogenous malonate.

REFERENCE TO PROVISIONAL APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/242,533 filed on Oct. 23, 2000, the entire disclosureof which is incorporated by reference herein.

INTRODUCTION

[0002] Malonyl-CoA is essential as a metabolic substrate in all livingorganisms studied so far and it also plays a role as a modulator ofspecific protein activity (for a review see Brownsey et al., 1997).Malonyl-CoA is a substrate for fatty acid synthase (FAS) (Bloch andVance, 1977), for polyketide synthases (PKS) in plants, fungi andbacteria (Hopwood & Sherman, 1990) and for fatty acid chain-elongationsystems (Saggerson, et al., 1992). Understanding the pathway(s) thatlead to the biosynthesis of malonyl-CoA in Streptomyces might have anoutstanding interest, since these micro-organisms are well known to havethe ability to synthesize a vast array of pharmaceutically importantpolyketide compounds (such as antibiotic, antiparasitic, antifungal,immunosuppressant and/or antitumour polyketides), where malonyl-CoA isused as the most common extender unit (Hopwood & Sherman, 1990).Therefore, information gained on the enzyme(s) involved in the supply ofthis key metabolite will be relevant, not only for a betterunderstanding of the primary metabolism of Streptomyces, but forimproving production of many useful secondary metabolites.

[0003] Biosynthesis of malonyl-CoA occurs in most species through theATP-dependent carboxylation of acetyl-CoA by an acetyl-CoA carboxylase(ACCase) (Bloch & Vance, 1977; Harwood, 1988). The overall reactioncatalyzed by ACCase is a two step process that involves ATP-dependentformation of carboxybiotin followed by transfer of the carboxyl moietyto acetyl-CoA. The importance of this biosynthetic pathway is mostdirectly reflected by the fact that ACCase expression is essential fornormal growth of bacteria (Perez, et al., 1998; Li and Cronan, 1993),yeast (Hasselacher, et al., 1993) and isolated animal cells in culture(Pizer, et al., 1996).

[0004] Several complexes with ACCase activity have been purified fromvarious actinomycetes. Interestingly, these complexes have also shownthe ability to carboxylate other substrates like propionyl- andbutyryl-CoA (Erfle, 1973; Henrikson and Allen, 1979; Huanaiti andKolattukudy, 1982). This property has led to these enzyme being calledacyl-CoA carboxylases, and all of them have been shown to consist of twosubunits, a larger one (α-chain) with the ability to carboxylate itscovalently bound biotin group, and a smaller sub-unit (β-chain) bearingthe carboxyl transferase activity. However, there is no informationgained, so far, regarding the physiological role of these enzymes.

[0005] In Streptomyces the purification of a complex with ACCaseactivity has proved to be unsuccessful, probably due to its highinstability (Bramwell et al., 1996). However ACCase activity has beenreadily measured in crude extracts of S. coelicolor (Bramwell et al.,1996; Rodríguez and Gramajo, 1999), indicating that this enzyme complexwas present in this micro-organism.

[0006] A pathway for the biosynthesis of malonyl-CoA in S. aureofacienshas been described that does not involve ACCase (Behal et al., 1977;Laakel et al., 1994). This route involves the anaplerotic enzymesphosphoenolpyruvate carboxylase and oxaloacetate dehydrogenase. In S.coelicolor A3(2), no evidence for the presence of oxaloacetatedehydrogenase has been found (Bramwell et al., 1993); thus, biosynthesisof malonyl-CoA in this organism seemed to occur exclusively through theACCase enzyme activity.

[0007] Attempts carried on in S. coelicolor to characterize enzymes withcarboxylase activity, have led to the characterization of two complexesexhibiting exclusively PCCase activity. The PCCase purified by Bramwellet al., (1996) comprises a biotinylated protein of 88 kDa, PccA, and anon-biotinylated component, the carboxyl transferase, of 66 kDa. Morerecently the inventors have also characterized at both the genetic andbiochemical levels, the components of a second PCCase in this bacterium.In vitro reconstitution experiments have shown that an active complexcould be obtained by mixing a carboxyl transferase component of 59 kDa(deduced MW, though it runs anomalously in SDS-PAGE, with an apparent MWof 65 kDa), PccB, with either of the two almost identical biotinylatedcomponents named AccA1 and AccA2 (Rodríguez and Gramajo, 1999). Recentlya gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoAsynthetase (MatB) and a putative decarboxylate carrier protein (MatC)has been proposed as the pathway for malonate metabolism in Rhizobiumtrifolii (An and Kim, 1998). After the transport of the malonate byMatC, the malonate is converted into malonyl-CoA by MatB and finallydecarboxylated to acetyl-CoA by MatA. However, the fact that the K_(m)of the malonyl-CoA decarboxylase for malonyl-CoA is relatively high hasled the inventors to propose that malonyl-CoA synthesised from malonateby malonyl-CoA synthetase (rather than malonyl CoA synthesised byACCase) is the major source for fatty acid biosynthesis in thebacterioid R. trifolii. Interestingly, genes with very high identity toMatC and MatB have been recently reported in the S. coelicolor genomeproject, suggesting that malonyl-CoA could also be synthesized frommalonate in this micro-organism.

[0008] The inventors have identified an essential acyl-CoA carboxylaseof S. coelicolor, and provide detailed genetic and biochemicalcharacterization. The enzyme complex contains a unique sub-unitcomposition and appears to be the main pathway for the biosynthesis ofmalonyl-CoA, one of the key metabolites in the linkage between primaryand secondary metabolism. An alternative pathway for the biosynthesis ofmalonyl-CoA is also proposed for cultures growing in malonate, and itmost probably involves the matB and matC homologues of R. trifolii.However, even in these growing conditions, the acyl-CoA carboxylaseseems to be essential for the viability of the micro-organism.

SUMMARY OF INVENTION

[0009] Two genes accB and accE, forming a single operon, have beencloned from Streptomyces coelicolor A(3)2. The deduced amino acidsequence of AccB showed high similarity to carboxyl transferase ofseveral propionyl- or acyl-CoA carboxylases of different actinomycetes.By contrast, AccE did not show any significant homology with proteinsequences deposited in the GenBank data base. Heterologous expression ofaccB and accE in Escherichia coli and in vitro reconstitution of enzymeactivity in the presence of the biotinylated component AccA1 or AccA2confirmed that AccB was the carboxyl transferase subunit of an acyl-CoAcarboxylase.

[0010] These experiments also established that AccE was a necessarycomponent to obtain a fully active enzyme complex, whose subunitcomposition seems to be unique within this type of carboxylase. Genedisruption experiments clearly determined that AccB was essential for S.coelicolor viability. This protein together with AccA2, a biotinylatedcomponent essential for the viability of this micro-organism (Rodríguezand Gramajo, 1999), are the best candidates to form an acyl-CoAcarboxylase, whose main physiological role is, most probably, thebiosynthesis of malonyl-CoA.

[0011] Transcriptional studies of accBE, accA2 and accA1 have shown thataccBE and accA2 are mainly expressed during vegetative and transitionphase of growth, although some expression of these genes also occurredduring stationary phase where they should provide the malonyl-CoAsubunits for secondary metabolites biosynthesis. accA1 is only expressedduring the transition phase of growth and its role in the formation of acarboxylase complex involved in providing the substrate for polyketidecompounds of S. coelicolor is discussed.

[0012] Finally, an alternative route for the biosynthesis of malonyl-CoAis proposed when malonate is used as a carbon source. However, thisroute seems unable to substitute the main one, determined by theacyl-CoA carboxylase.

[0013] Accordingly, in a first aspect, the present invention provides anucleic acid comprising a nucleic acid sequence which encodes an AccBpolypeptide and/or an AccE polypeptide, or a nucleic acid sequencecomplementary thereto.

[0014] In a second aspect, the present invention provides a nucleic acidcomprising a nucleic acid sequence which encodes an AccA1 and/or AccA2polypeptide, or a nucleic acid sequence complementary thereto. It isbelieved that such nucleic acid was not made available to the publicbefore Oct. 24, 1999, when the amino acid sequences of thesepolypeptides were disclosed in an oral presentation.

[0015] Exemplary nucleic acid sequences encoding the AccB, AccE, AccA1and AccA2 polypeptides are given herein. Preferred embodiments of theinvention include such sequences. However, it would be a matter ofroutine for the skilled person to obtain other nucleic acid sequencesencoding these polypeptides, e.g. by introducing mutations which do notalter the encoded amino acid sequence, by virtue of the degeneracy ofthe genetic code, or by introducing mutations which alter the encodedamino acid sequence, within limits as defined below. Moreover, nucleicacids encoding variants of the polypeptides may be obtained e.g. byscreening different strains of S. coelicolor or closely related speciesof Streptomyces using degenerate probes based on the sequences givenherein.

[0016] Preferred nucleic acids of the first and second aspects encodeAccB and AccE polypeptides along with an AccA1 and/or an AccA2polypeptide (preferably AccA2).

[0017] The nucleic acid sequences encoding Acc polypeptides arepreferably in operative association with regulatory sequences, e.g.sequences which enable constitutive or inducible expression inStreptomyces species. Examples of plasmids which include such regulatorysequences and of suitable promoters are given herein. A suitableinducible promoter is tipA (inducible by thiostrepton); suitableconstitutive promoters are ermE and the optimised ermE*. Alternatively,naturally occurring nucleic acid sequences may be in operativeassociation with the regulatory sequences with which they are normallyassociated, or corresponding regulatory sequences from homologous genesin other strains or species. For example, the nucleic acid sequences maybe in operative association with the corresponding regulatory (e.g.promoter) sequences defined herein.

[0018] For detailed protocols relevant to this and other aspects, seestandard reference texts, such as Sambrook et al. (1989) and Hopwood etal. (1985).

[0019] In a third aspect, the present invention separately providesAccB, AccE, AccA1 and AccA2 polypeptides having amino acid sequencesencoded or encodable by the respective nucleic acid sequences referredto in the first and second aspects.

[0020] In a fourth aspect, the present invention provides: vectorscontaining the nucleic acids of the first and second aspects (preferablyvectors, e.g. plasmids, suitable for transforming Streptomyces speciesfor expression therein) and cells, particularly Streptomyces cells,transformed with such vectors. Furthermore, the present inventionprovides a method of producing a secondary metabolite of a Streptomycesspecies, the method comprising culturing such transformed Streptomycescells and extracting the secondary metabolite from the cell culture. Themetabolite may be purified and/or formulated as a commercial productaccording to standard procedures.

[0021] In a fifth aspect, the invention provides a method of modifying asecondary metabolite-producing strain of a Streptomyces species toincrease production of said secondary metabolite, the method comprisingmodifying said strain to express, or to increase expression of, nucleicacid encoding one or more polypeptides selected from the groupconsisting of AccB, AccE, AccA1 and AccA2.

[0022] In a sixth aspect, the present invention provides a method ofmodifying a strain of a Streptomyces species to increase ACCase and/orPCCase activity, the method comprising modifying said strain to express,or to increase expression of, nucleic acid encoding one or morepolypeptides selected from the group consisting of AccB, AccE, AccA1 andAccA2.

[0023] In a seventh aspect, the present invention provides a modifiedstrain of a Streptomyces species, produced or producible according tothe method of the fifth or sixth aspect. Also provided are cells of saidstrain, methods of producing secondary metabolites comprising culturingsaid cells and extracting the secondary metabolite, which may bepurified and/or formulated as a commercial product.

[0024] In an eighth aspect, the invention provides a method ofincreasing production of a secondary metabolite in cells of aStreptomyces species, the method comprising culturing said cells in thepresence of exogenous malonate, preferably at a concentration of atleast about 0.1%, more preferably at least about 0.2%, 0.4%, 0.5%, 0.75%or 1%, though higher concentrations may be used. 1% represents 1 g per100 ml of medium.

DETAILED DESCRIPTION

[0025] In relation to the fifth and sixth aspects, the modificationpreferably provides for increased expression of nucleic acid encodingmore than one of AccE, AccE, AccA1 and AccA2, more preferably at leastAccB and AccE or at least AccB and either AccA1 or AccA2, morepreferably AccB, AccE and either AccA1 or AccA2. Of AccA1 and AccA2,AccA2 is preferred. Increased expression of nucleic acid encoding bothAccA1 and AccA2 (usually in combination with AccB and optionally AccE)is also contemplated.

[0026] The methods of the fifth and sixth aspects preferably include astep of transforming a Streptomyces cell with a said nucleic acid underthe control of a constitutive or inducible promoter, preferably a strongpromoter. However, the expression of existing said nucleic acid could beincreased, e.g. by placing them under the control of a stronger promotersequence or sequences.

[0027] Exogenous said nucleic acid can replace existing said nucleicacid in the cell, or can be added without removing or functionallydeleting existing said nucleic acid.

[0028] Acc Polypeptides and Acc Genes

[0029] In the definitions herein of the invention, and of the scope ofprotection (but not, except where the context requires otherwise, in theexperimental sections), the term AccB is intended to include not only apolypeptide having the deduced amino acid sequence encoded by thenucleic acid sequence of FIG. 12 (though this is a preferredembodiment), but also a polypeptide which is a variant (e.g. an allelicor isoallelic variant) or a derivative of said polypeptide, having atleast about 60% amino acid identity with said polypeptide, preferably atleast about 65%, 70% or 75%, especially preferably (in view of thesimilarity of AccB as disclosed herein to another protein of unconfirmedfunction) at least about 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99%identity. Such a variant or derivative may possess any one or more ofthe biological properties of the wild-type AccB protein, as disclosedherein, e.g. complex formation with AccA1, AccA2 and/or AccE (orallosteric regulation by AccE), ACCase and/or PCCase activity when AccBis co-expressed with AccA1, AccA2 and/or AccE, or increased secondarymetabolite production when AccB is overexpressed in Streptomyces species(preferably in conjuction with AccA1, AccA2 and/or AccE).

[0030] Similarly, the term AccE is intended to include not only apolypeptide having the deduced amino acid sequence encoded by thenucleic acid sequence of FIG. 13 (though this is a preferredembodiment), but also a polypeptide which is a variant (e.g. an allelicor isoallelic variant) or a derivative of said polypeptide, having atleast about 40% amino acid identity with said polypeptide, preferably atleast about 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity. Such avariant or derivative may possess any one or more of the biologicalproperties of the wild-type AccE protein, as demonstrated herein, e.g.complex formation with AccA1, AccA2 and/or AccB (or allostericregulation of AccB), ACCase and/or PCCase activity when AccE isco-expressed with AccB, or increased secondary metabolite productionwhen AccE is overexpressed in Streptomyces species (preferably inconjuction with AccB).

[0031] Similarly, the terms AccA1 and AccA2 are intended to include notonly the polypeptides having the amino acid sequences shown in FIG. 11(though these are respective preferred embodiments), but alsopolypeptides which are variants (e.g. allelic or isoallelic variants) orare derivatives of said polypeptides, having at least about 75% aminoacid identity with said polypeptide, preferably at least about 80%, 85%,90%, 92%, 94%, 96%, 98% or 99% identity. Such variants or derivativesmay possess any one or more of the biological properties of thewild-type AccA1 or AccA2 polypeptides, as disclosed herein, e.g. complexformation with AccB and/or AccE, ACCase and/or PCCase activity whenAccA1 or AccA2 is co-expressed with AccB and/or AccE, or increasedsecondary metabolite production when AccB is overexpressed inStreptomyces species (preferably in conduction with AccB and/or AccE).

[0032] A variant or a derivative of a given peptide may have one or moreof internal deletions, internal insertions, terminal truncations,terminal additions, or substitutions of one or more amino acids,compared to the given peptide.

[0033] References to nucleic acid encoding AccA1, AccA2, AccB and/orAccE (or to accA1, accA2, accB and/or accE genes) should be interpretedaccordingly.

[0034] In relation to the first aspect, preferred nucleic acids comprisea nucleic acid sequence having at least about 50%, preferably at leastabout 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% nucleic acid sequenceidentity with the accB nucleic acid sequence shown in FIG. 12. Otherpreferred nucleic acids comprise a nucleic acid sequence having at leastabout 40%, preferably at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%,98% or 99% nucleic acid sequence identity with the accE nucleic acidsequence shown in FIG. 13. Similarly, in relation to the second aspect,preferred nucleic acids comprise a nucleic acid sequence having at leastabout 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%or 99% nucleic acid sequence identity with the accA1 or accA2 nucleicacid sequence shown in FIG. 11.

[0035] Secondary Metabolites and Streptomyces Species

[0036] While the experimental disclosure herein relates to theproduction of Act (actinomycin) and Red (undecylprodigiosin) in S.coelicolor A3(2) (strain M145), it is thought that the teaching isapplicable to other strains of Streptomyces in particular, it is thoughtthat overexpession of all three Acc polypeptides (i.e. AccB, AccE andAccA1 and/or AccA2) will lead to increased malonyl-CoA production insubstantially any Streptomyces species or even in other actinomycetes orin fungi (which also produce polyketide compounds). Since malonyl-CoA isan essential metabolic substrate, it is thought that this will lead togreater yield of desired secondary metabolites (for which see page 1),e.g. polyketides (including antibiotic polyketides) and fatty acids.

[0037] Preferred secondary metabolites are, however, antibiotics,especially Act and Red.

[0038] Preferred Streptomyces species are the closely related species S.coelicolor, S. violaceoruber, S. lividans and S. parvulus, especially S.coelicolor. Strains of such species are commonly available, e.g. fromthe ATCC, for example under ATCC deposit numbers 12434 for S. parvulusand 19832 for S. violaceoruber. S. coelicolor A3(2) and S. lividans 66are available from the John Innes Culture Collection (Norwich, UK) underJICC deposit numbers 1147 and 1326, respectively. However, the inventionis not limited to such particular strains.

[0039] Acetyl-CoA

[0040] In preferred embodiments, present invention further provides forthe increased production in Streptomyces of acetyl-CoA, since it isthought that when ACCase activity is increased by the methods and meansof the present invention, production of malonyl-CoA may become limitedby the availability of the substrate acetyl-CoA. It is proposed thatincreased acetyl-CoA production could then lead to a further increasedrate of malonyl-CoA production and hence secondary metaboliteproduction. For example, oils or fatty acids could be used as the carbonsource (together with glucose); fatty acids are degraded by b-oxidationgiving high levels of acetyl-CoA.

[0041] Sequence Identity

[0042] “Percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the sequence with which it isbeing compared, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. The % identity values used herein are generated by WU-BLAST-2which was obtained from Altschul et al. (1996);http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, most of which are set to the default values. The adjustableparameters are set with the following values: overlap span=1, overlapfraction=0.125, word threshold (T)=11. The HSPS and HSPS2 parameters aredynamic values and are established by the program itself depending uponthe composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values may be adjusted to increase sensitivity. A% amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region, multiplied by 100. The“longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-BLAST-2 to maximize the alignmentscore are ignored).

[0043] “Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues in the sequence under comparison.The identity values used herein were generated by the BLASTN module ofWU BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

[0044] Culture and Purification

[0045] Methods of genetic manipulation, cell culture and purification ofexpression products produced in cell culture are well known to theskilled person, e.g. from standard textbooks such as Sambrook et al(1989). In particular, methods for genetically manipulatingStreptomyces, culturing Streptomyces under conditions suitable forsecondary metabolite (e.g. polyketide and/or antibiotic production) andpurifying secondary metabolites from Streptomycete cell culture mediumare well known, e.g. from Hopwood et al. (1985) and Kieser et al (2000).

[0046] Formulation

[0047] Similarly, methods of formulating active compounds (e.g.polyketides, particularly antibiotics) as pharmaceuticals are well knownin the art. Such pharmaceutical formulations may comprise, in additionto the active compound, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,transdermal, transmucosal, intramuscular, intraperitoneal routes.

[0048] Suitable carriers, adjuvants, excipients, etc. can be found instandard pharmaceutical texts, for example, Remington's PharmaceuticalSciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; andHandbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0049] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0050] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active compound will be in theform of a parenterally acceptable aqueous solution which is pyrogen-freeand has suitable pH, isotonicity and stability. Those of relevant skillin the art are well able to prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilisers,buffers, antioxidants and/or other additives may be included, asrequired.

[0051] Formulations suitable for transmucosal administration includeliquids, solutions, suspensions, emulsions, suppositories, pessaries,gels, pastes, ointments, creams, lotions, oils, as well as patches,adhesive plasters, depots, and reservoirs.

[0052] Formulations suitable for transdermal administration includegels, pastes, ointments, creams, lotions, and oils, as well as patches,adhesive plasters, bandages, dressings, depots, and reservoirs.

[0053] Ointments are typically prepared from the active compound and aparaffinic or a water-miscible ointment base.

[0054] Creams are typically prepared from the active compound and anoil-in-water cream base. The aqueous phase of the cream base may includeat least about 30% w/w of a polyhydric alcohol, i.e., an alcohol havingtwo or more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active compound through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide and related analogues.

[0055] Formulations may suitably be provided as a patch, adhesiveplaster, bandage, dressing, or the like which is impregnated with one ormore active compounds and optionally one or more other pharmaceuticallyacceptable ingredients, including, for example, penetration, permeation,and absorption enhancers.

[0056] Administration is preferably in a “prophylactically effectiveamount” or a “therapeutically effective amount” (as the case may be,although prophylaxis may be considered therapy), this being sufficientto show benefit to the individual. The actual amount administered, andrate and time-course of administration, will depend on the nature andseverity of what is being treated. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors, and typically takes account ofthe disorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of the techniques and protocols mentionedabove can be found in Remington's Pharmaceutical Sciences (supra).

[0057] A pharmaceutical formulation may be administered alone or incombination with other treatments, either simultaneously or sequentiallydependent upon the condition to be treated.

[0058] The work underlying the invention will now be described indetail, by way of example only, with reference to the accompanyingfigures.

FIGURES

[0059]FIG. 1 Organization of the genomic region of S. coelicolor M145chromosome harbouring accB and accE genes. A. Genetic and physical mapof the 6.2 kb insert in pRM08. The secondary structure downstream accErepresents a rho-independent transcriptional terminator. Fragments I andII were amplified by PCR with the pair of oligos accBup-accBdown andaccBEup-accBEdown respectively, uniquely labelled at the 5′-end (*) andused as probes in transcriptional analysis of the accBE operon. B.Physical map of the DNA fragments cloned in pET22b(+) and used for theheterologous expression of accB and/or accE. Only the most relevantrestriction sites are shown: B, BamHI; Bc, BclI; E, EcoRI; K, KpnI; Nd,NdeI; N, NotI; S, SpHI.

[0060]FIG. 2 Attempted disruption of accB. A. Diagram showing theintegration of pTR124 through one of the accBE flanking regions and theresolution of the cointegrate by a second event of homologousrecombination. The crossed out arrow indicates the impossibility ofobtaining the replacement of the wild-type accB by the Hyg^(R) mutantallele. B. The integration of a second copy of the accBE genes in theΦC31 att site of T124 (to yield strain T149) allowed the replacement ofthe wild-type accB by a mutant allele containing the Hyg resistancecassette.

[0061]FIG. 3 Growth-phase dependent expression and transcription startsite of the accBE operon. A. S1 nuclease mapping of accB, actII-ORF4 andhrdB, using RNA isolated from a liquid time course of S. coelicolorM145. Exp, Trans and Stat indicate the exponential, transition andstationary phase of growth, respectively. B. The nucleotide sequences ofboth strands from the accB promoter region are shown. The arrowindicates the most likely transcription start point for the accBEpromoter, as determined by S1 nuclease mapping. The potential -10 and-35 regions for the accBEp are underlined. C. S1 nuclease mapping of theaccB accE intergenic region using a 563 nt probe. FLP represents thefull-length RNA-protected fragment that is 13 nt shorter than the probe.

[0062]FIG. 4 Growth-phase dependent expression of accA2 and accA1. S1nuclease mapping of accA2 (A) and accA1 (B), using RNA isolated from aliquid time course of S. coelicolor M145.

[0063]FIG. 5 Mapping of the accA2 and accA1 transcription start point.A. High resolution S1 nuclease mapping of the 5′end of the accA2transcript. S1, RNA-protected products of the S1 nuclease protectionassay. Lanes labelled A, C, G and T indicate a dideoxy sequencing ladderusing the same oligonucleotide that was used to make the S1 probe(accA2down). B. High resolution S1 nuclease mapping of the 5′ end of theaccA1 transcript. S1, RNA-protected products of the S1 nucleaseprotection assay. Lanes labelled T, G, C and A indicate a dideoxysequencing ladder using the same oligonucleotide that was used to makethe S1 probe (accA1down). C. Sequence of the accA2 and accA1 upstreamregions, indicating the most likely transcription start points for thepromoters of each of the accA1 and accA2 genes (bent arrows). Thepotential -10 and -35 sequences for the accA1 and accA2 promoters areunderlined. The potential ribosomal binding sites (rbs) are highlightedwith bold letters. The 16 nt direct repeats (DR) found upstream of thetranscription start point of accA1p1 are indicated with straight arrows.

[0064]FIG. 6 Construction and analysis of the accBE conditional mutant.A. Diagram showing the integration of pIJ8600 in strain M86 and theexpected organisation of the Campbell integration of pTR94 in M94.Restriction sites: B. BamHI; N, NotI; Nd, NdeI;

[0065] S, SacI; Sp, SphI; Xb, XbaI. B. Hybridisation analysis ofSouthern blot of SacI-digested DNAs from M145, M86 and M94. The probewas the internal NdeI-XbaI fragment of accB shown in A (see FIG. 10).

[0066]FIG. 7 Expression of the acyl-CoA components in M86 and M94. A.SDS-PAGE of cell-free extracts of S. coelicolor M86 and M94 strainsgrown in YEME medium containing 10 μg/ml Am with or without the additionof 5 μg/ml Th. B. A duplicate of the SDS-PAGE gel shown in A wassubjected to Western blotting and stained for biotinylated proteins byusing alkaline phosphatase-streptavidin conjugate.

[0067]FIG. 8A Growth curves of M145, M86 and M94 strains. 10⁸ spores ofstrains M86 and M94 were inoculated in YEME medium containing 10 μg ofAm or 10 μg/ml Am and 5 μg/ml of Th. 10⁸ spores of M145 were inoculatedin YEME. The growth was followed by measuring OD_(450 nm).

[0068]FIG. 8B Actinorhodin production in M94 and M145 in cultures grownin the presence of 5 μg of Th.

[0069]FIG. 9 Morphological and physiological differentiation of M86 andM94 in the presence of Th. Spores of M86 and M94 were spread in R2 or R5medium containing 10 μg/ml Am. A drop containing 1 μg of Th was spottedin the centre of each plate. The picture shows the results obtainedafter the incubation of the plate at 30° C. for 48 h.

[0070]FIG. 10 The sequence of the amplification product obtained fromaccB using primers TC16 and TC17. NdeI (CATATG) and XbaI (TCTAGA) sitesintroduced into the accB by the primers are shown in bold. The 1 kbNdeI-XbaI fragment was cloned into pIJ8600.

[0071]FIG. 11 A. Amino acid sequences and B. Nucleic acid sequences ofaccA1 and accA2.

[0072]FIG. 12 A. Amino acid sequence and B. Nucleic acid sequence ofaccB.

[0073]FIG. 13 A. Amino acid sequence and B. Nucleic acid sequence ofaccE.

[0074]FIG. 14 Plasmid map for the construction of an expression vectorfor accA, accB and accE.

EXAMPLE 1 Cloning of AccBE Genes

[0075] pccB of S. coelicolor (Rodríguez and Gramajo, 1999) was used asan heterologous probe in Southern blot experiments. When a BamHI digestof S. coelicolor DNA was probed with pccB and washed under low stringentconditions, a second, low hybridising, band was readily detected (datanot shown). The target sequence was cloned from a size-enriched libraryas a 2.5 kb BamHI fragment and sequenced as described in ExperimentalProcedures (below). The sequence revealed the presence of an incompleteORF with high homology to pccB. The complete gene was finally cloned asa 6 kb SstI fragment yielding pRM08 (FIG. 1). Sequencing and analysis ofthis DNA fragment revealed the presence of an ORF that exhibitedend-to-end similarity with a putative decarboxylase (though the realfunction is unknown) of S. cyanogenous (Westrich et al., 1999), with theS. coelicolor PccB (Rodríguez and Gramajo, 1999) and with the β-subunit(PccB) of the Sac. erythraea PCCase (Donadio, et al., 1996). The levelsof identity were 76%, 57% and 56%, respectively. The gene encoding thisnew putative carboxyl transferase was called accB.

[0076] Surprisingly, the sequence also revealed the presence of a smallORF, designated accE, whose start codon is only 17 bp downstream of thetermination codon of accB. A 17 nt inverted repeat, which could functionas a factor-independent bidirectional transcriptional terminator(reviewed in Lewin, 1994), separates accE from three convergent ORFswith homology to putative proteins of M. tuberculosis with unknownfunctions. The putative AccE polypeptide has a deduced molecular mass of7.07 kDa and no significant homology to this polypeptide was found in asearch of sequences deposited in the GenBank database.

[0077] Upstream of accB there is an ORF highly homologous to severalknown hialuronidases.

EXAMPLE 2 AccB is Essential for S. coelicolor Viability

[0078] An accB mutant was constructed by gene replacement (FIG. 2A). AHyg-resistant cassette was cloned in the unique BamHI site present inthe coding sequence of accB, contained in PTR80. After an intermediateconstruction in pIJ2925, a BglII fragment containing the mutated allelewas finally cloned in the conjugative vector pSET151. The resultingplasmid, pTR124, was cloned into the E. coli donor strainET12567/pUZ8002 and transferred by conjugation into M145. Exconjugantswere selected for Th^(R) Hyg^(R) for a simple crossover event. One ofthe exconjugants, named T124, was taken through four rounds ofnon-selective growth (SFM Hyg) to promote homologous recombination forthe second crossover. Spores were plated to give single colonies andseveral thousands screened for Th sensitivity (which would havereflected successful gene replacement), but no Th^(S) isolates wereobtained. This result suggested that accB is essential for S. coelicolorviability.

[0079] The inventors proposed, however, that if a second copy of accBwere present in the chromosome of T124, a second crossover event(leading to the replacement of the wild type gene by the Hyg^(R) mutantallele) would then be allowed. To confirm this hypothesis, pTR149, whichcontains a copy of the accBE genes under its own promoter (seeExperimental procedures, FIG. 2B), was first integrated in the ΦC31 attBsite of T124. (The introduction of a second copy of both genes into thechromosome was prompted by the probability of a polar effect on accEtaking place after the gene replacement event and because AccE isimportant for the recovery of a fully active acyl-CoA carboxylasecomplex—see in vitro reconstitution experiments below). The resultantstrain T149 (Hyg^(R), Th^(R), Am^(R)) was passed through three rounds ofsporulation on SFM Hyg Am and after the screening of approximately 500colonies, 20 were found to be Am^(R) Hyg^(R) Th^(S). The finalchromosomal organization of accB in each of the strains constructed(T124, T149 and T149A), was analyzed by Southern blots using an internalfragment of accB as a probe.

EXAMPLE 3 Heterologous Expression of AccB, AccE and In VitroReconstitution of an Acyl-CoA Carboxylase Complex

[0080] Since accB proved to be essential for S. coelicolor viability, wecould not clearly evaluate in vivo the physiological function of thisgene product.

[0081] In order to study if AccB and AccE were components of an acyl-CoAcarboxylase complex, we attempted in vitro reconstitution of the enzymeactivity by mixing E. coli cell-free extracts containing the AccB andAccE with cell-free extracts containing the biotinylated sub-units AccA1or AccA2. E. coli does not contain an ACCase enzyme, so ACCase activitycannot be assayed directly by carboxylation of acetyl-CoA (Polakis etal., 1972); therefore, the acyl-CoA carboxylase activity measured inthese crude extracts exclusively represents the activity of theheterologous complexes reconstituted in vitro.

[0082] Heterologous expression of accB and accE was attempted byintroducing a NdeI site at the ATG start codon of accB; after anintermediate construction (see Experimental procedures), accBE wascloned as a NdeI-SacI fragment into pET22(b), yielding pTR88 (FIG. 1).Transformation of E. coli BL21(DE3) with this plasmid yielded strain RG8(Table 1). Crude extracts of RG8, prepared from IPTG-induced cultures,showed a clear over-expression of a 64 kDa protein in a 15% SDS-PAGE,corresponding to AccB; by contrast, AccE was not clearly visualised byCoomassie blue staining of the same gel (data not shown). In vitroreconstitution of an acyl-CoA carboxylase was then attempted my mixingcrude extracts prepared from IPTG-induced cultures of RG8 with cell-freeextracts of the E. coli strains RG7, which overproduces the biotinylatedprotein AccA1. After incubation for 1 h at 4° C., the mixture wasassayed for ACCase and PCCase activity. As shown in Table 2 an enzymecomplex showing high levels of both ACCase and PCCase activities wassuccessfully reconstituted

[0083] To study if cell-free extracts containing AccB but not AccE werecapable of reconstituting an active acyl-CoA carboxylase complex whenmixed with cell-free extracts containing AccA1, we constructed a newpET22(b) derivative that only expresses accB. For this we took advantageof the NotI site present approximately in the middle of the codingsequence of accE and cloned the NdeI-NotI fragment from pTR88 into theexpression vector, yielding pTR90 (FIG. 1).

[0084] Cell-free extracts of RG9, obtained by transformation ofBL21(DE3) with pTR90, showed high levels of soluble AccB after IPTGinduction. However, the acyl-CoA carboxylase complex reconstituted invitro, after mixing cell-free extracts of RG9 (AccB) and RG7 (AccA1),showed much lower levels (approximately 10%) of ACCase and PCCaseactivities than the acyl-CoA carboxylase previously obtained by mixingRG8 with RG7 cell-free extracts (Table 2). Since the levels of AccB incell-free extracts of RG8 and RG9 were essentially the same, we inferredfrom these experiments that AccE was necessary in order to obtain afully active acyl-CoA carboxylase complex.

[0085] To confirm that the absence of AccE was the responsible of thelower acyl-CoA carboxylase activities, we studied the effect that theaddition of cell-free extract containing AccE, had on the crude extractscontaining AccB and AccA1 proteins. For this we first constructed strainRG10 (BL21(DE3) containing pTR107) that expresses high levels of solubleAccE (data not shown).

[0086] When cell-free extracts of RG10 where mixed with those of RG9(AccB) and RG7 (AccA1) and incubated for 1 h on ice, the levels ofenzyme activity where at least five times higher than in the controlexperiment, without the addition of AccE (Table 2). Although the resultspresented in this section clearly show that AccE is a functional part ofthe acyl-CoA carboxylase, enzyme kinetics studies with purifiedcomponents will be necessary to understand more precisely the role ofthis protein in the enzyme complex activity. Similar results wereobtained in all the reconstitution experiments mentioned above whenAccA1 was replaced by AccA2 as the biotinylated component of theacyl-CoA carboxylase, indicating that either AccA1 or AccA2 can beefficiently used as the α-subunit of this enzyme complex.

EXAMPLE 4 Transcriptional Analysis of AccBE, AccA1 and AccA2

[0087] At least four combinations that resulted in active carboxylasecomplexes have been reconstituted by mixing the β-subunits PccB(Rodríguez and Gramajo, 1999) or AccB (this work) with either of the twoalmost identical α-subunits, AccA1 or AccA2. In any of these complexesthe carboxyl transferase subunit seems to dictate the substratespecificity; thus, PccB seems to recognize only propionyl-CoA, whileAccB has a broader substrate specificity, which allows the enzyme torecognize either acetyl- or propionyl-CoA. Moreover, a third complexwith PCCase activity has also being described in S. coelicolor(Bramwell, et al., 1996). These findings show a remarkable overlappingof gene function in Streptomyces species. We followed two differentapproaches to gain more information on this; one was the generation ofmutants and the second the study of the mRNA levels of some of thesefour genes throughout the different growth stages by using S1 nucleaseprotection.

[0088]S. coelicolor A3(2) strain M145 was grown in SMM medium and RNAextracted at exponential, transition and stationary phase. S1 nucleaseprotection of accB was performed by using a 483 bp PCR product, uniquelylabelled at the 5′end of the downstream oligo. Transcription of accBoccurs primarily during active growth (exponential and transitionphases), while its level of expression decayed significantly afterentering into stationary phase (FIG. 3A). The transcripts of the majoressential sigma factor hrdB and of the pathway-specific activator genefor acitnorhodin biosynthesis, actII-ORF4, were also studied as positivecontrols for the RNAs used in these experiments. As expected fromprevious results, hrdB was expressed constantly throughout growth(Buttner, M. J., 1990), while actII-ORF4 had a peak of expression duringtransition phase that shut off in stationary phase (Gramajo, et al.,1993).

[0089] The RNA-protected fragments found for accB corresponded to atranscription start site 1 bp upstream, or in the adenine, of the mostlikely translation start site of accB. Upstream of the transcriptioninitiation site we found a putative -10 and -35 promoter regions with ahigh consensus sequences of promoters recognised by the vegetativeσ^(hrdB) (Strohl, 1991) (FIG. 3B).

[0090] In order to find out if accB and accE were co-transcribed as aunique bi-cistronic mRNA, a new 563 bp probe was obtained by PCR. Forthis we used a 5′oligo corresponding to a sequence within the codingregion of accB and a 3′oligo corresponding to a sequence within accE.The full-length RNA-protected fragment was easily differentiated fromthe probe-probe re-annealing due to the addition of a 13 nt tail to the5′oligonucleotide (Experimental Procedures). The results obtained inthis experiment clearly showed that accB and accE were part of the sametranscript, confirming that these two genes form a single-copy operon(FIG. 3C). Moreover, the expression of accBE during the different growthphases as detected with this new probe followed the same profile as theexpression observed with the probe used for accB.

[0091] The levels of accA2 and accA1 mRNA present throughout growth werealso studied by S1 protection experiments (FIG. 4). The probe used foraccA2 was a 766 bp DNA fragment generated by PCR and uniquely labelledon the 5′end of the oligo corresponding to the sequence within accA2.This experiment showed the existence of three mRNA-protected fragments.The growth phase-dependent expression of two of them, accA2p1 andaccA2p2, resemble very much that of the accBE operon. Thus, a constantand high level of expression occurs during exponential and transitionphase (TP), while the transcription shuts down when the cultures reachstationary phase (FIG. 4A).

[0092] Considering that the nucleotide sequences of accA1 and accA2 areidentical from the first two nucleotides upstream of the most probableGTG translation start sites down to the end of the probe (Rodríguez andGramajo, 1999), it is important to note that a fragment of 185 bp of theaccA2 probe could also be protected by the accA1 mRNA. Since the lowestRNA-protected fragment observed in FIG. 4A shows a different pattern ofexpression with respect to accA2p1 and p2, and considering that the sizeof the band corresponds to a 185 bp fragment, we believe that this bandmight represent the level of expression of accA1 (although we cannotrule out the existence of a third promoter for accA2, regulated in adifferent manner).

[0093] S1 nuclease protection of accA1 mRNA was performed by using a 563bp PCR product, uniquely labelled at the 5′end of the downstream oligo,corresponding to a sequence within accA1. As shown in FIG. 4B, theexpression of this gene occurs from at least three different putativepromoters, and all of them showed a clear burst of expression during thefirst hours of the TP, which rapidly shut down during late TP. Thispattern of transcription resembled very much the one observed for thethird RNA-protected band found for accA2. The transcription starts sitesfor the accA2p1 and p2 were mapped by high resolution S1 mapping (FIGS.5A and B). The transcription start points and the putative -10 and -35promoter regions of these two promoters are shown in FIG. 5C. A certaindegree of homology was found between the -10 consensus sequence ofaccA2p1 and p2 and the promoters recognised by the vegetative σ^(hrdB)(Strohl, 1992). High resolution S1 mapping of accA1 revealed that thetranscription start point of the most abundant mRNA species starts 88 bpupstream of the GTG initiation codon of AccA1 and the putative -10regions resemble, in some extent, the consensus sequences of promotersrecognised by σ^(hrdB). Interestingly, two direct repeat (DR) sequencesof 16 bp, containing only two mismatches, were found flanking theputative -35 region of accA1p1 and the transcription start point ofaccA1p2 (FIG. 5C). These DRs could represent DNA binding sitesrecognised by a putative regulator. A third putative promoter, accA1p3,was also detected in longer exposures and the most probable nucleotidestart sites are also indicated in FIG. 5C.

EXAMPLE 5 AccBE Genes are Essential in the Presence of Malonate

[0094] The presence of MatC and MatB homologues in S. coelicolorsuggested that this micro-organism was potentially capable oftransporting malonate within the cell through the MatC transporter, andthen activating malonate to malonyl-CoA with the putative malonyl-CoAsynthetase MatB. To test whether S. coelicolor was able to utilizemalonate as a sole carbon and energy source, we grew S. coelicolor in amodified SMM medium with no casamino-acids and containing 0.4% malonateinstead of glucose as a sole carbon source. In this medium S. coelicolorM145 was able to grow, indicating that MatC and MatB could be theproteins involved in the transport and activation of malonate tomalonyl-CoA, and suggesting that a decarboxylase that could convertmalonyl- into acetyl-CoA should also be present in this bacterium, toallow the use of malonate as a carbon and energy source.

[0095] This result encouraged us to test whether this route could alsobe an alternative pathway to provide malonyl-CoA to the cell. To provethis hypothesis we tried to obtain an acyl-CoA carboxylase minus mutantin the presence of malonate. For this we took spores of strain T124 andgrew them in liquid MM containing 0.4% of malonate instead of glucose.After 36 h of growth we sonicated the mycelia and spread them in SFMmedium containing 0.4% of malonate and incubated until sporulation.Spores were collected and treated in the same way one more time.Finally, spores harvested after the second round of sporulation werediluted out, inoculated in SFM malonate to give aprox. 500 colonies perplate and replica plated in SFM medium with or without Th. Afteranalyzing approximately 5000 isolated colonies, no Th^(S) were obtained.This result indicates that although malonate can be efficiently used asa sole carbon and energy source, the pathway involved in its catabolismcan not fulfill the malonyl-CoA requirements of the cell.

EXAMPLE 6 Construction of a Strain with the AccBE Operon Under theControl of a tipA Promoter

[0096] As shown above, the accBE operon, which encodes thecarboxyl-transferase and a previously unidentified ε sub-unit of anacyl-CoA carboxylase, is essential for the viability of S. coelicolorA3(2). In order to regulate the expression of this operon and study itseffect on the physiology of this microorganism, we constructed aconditional mutant strain where the expression of the accBE operon wasunder the control of the thiostrepton-inducible tipA promoter (Murakami,et al., 1989).

[0097] A 947 bp fragment containing a modified 5′end of the accB genewas cloned under the tipA promoter in pIJ8600 (Sun et al (1999) supra)to yield pTR93. After removal of the ΦC31 integration components (attand int) present in pTR93 we obtained pTR94, which was transformed intothe E. coli strain ET12567/pUZ8002 (MacNeil et al (1992)/Paget et al(1999)). Conjugation of pTR94 into the S. coelicolor strain M145 gaveseveral exconjugants Th^(R). One of these exconjugants, designated M94,was purified in SFM medium for further analysis. Integration of pTR94could only take place by Campbell recombination through the accBEhomologous sequences, and this event should leave a complete copy of theaccBE operon under the tipA promoter (FIG. 6A). To confirm that thisevent had occurred in M94, we performed Southern blot experiments of DNAsamples prepared from strains M145, M94 and M86. The last strain (M86)was obtained by integration of pIJ8600 in the ΦC31 att site of thechromosome and used as the best isogenic control for M94 (FIG. 6A). Asshown in FIG. 6B, a SacI digested DNA from M145 and M86 lights up aunique hybridisation band of 5.94 kb that contains the accBE operon. DNAfrom M94, instead, lights up two hybridising bands corresponding to theexpected sizes for the integration of pTR94 in the accBE operon (FIGS.6A and B).

EXAMPLE 7 Acyl-CoA Carboxylase Levels in M94 and M86

[0098] Cultures of the conditional accBE mutant M94 grew normally inYEME medium containing 5 μg of Th. Interestingly, in the absence of theantibiotic, the cultures were still able to grow, although at much lowerrate. This experiment re-confirms the leakiness of the tipA promoter (M.J. Bibb, personal communication). In order to determine the levels ofthe acyl-CoA carboxylase in conditions of induction or non-induction wecarried out the following protocol. YEME medium containing 10 μg of Amwas inoculated with spores of M94 (or M86) to give and initialOD₄₅₀=0.1. Cultures were grown for 12 h at 30° C. and after that time 5μg of Th was added to a half of each culture, keeping the other half asa control. Both flasks were then incubated for additional 24 h at 30° C.The harvested mycelia were disrupted by sonication and cell debrisremoved by centrifugation. Cell-free extracts were finally analysed bySDS-PAGE and used for enzyme assays. FIG. 7A shows a 60 kDa protein thatis only induced in cultures of M94 grown in the presence of Th; the sizeof this protein corresponded to the molecular mass of AccB. We were notable to detect an inducible band corresponding to AccE. The levels ofthe biotinylated components (AccA1 or AccA2) of the acyl-CoAcarboxylase, in each of the cell-free extracts, were analysed by amodified Western Blotting procedure (FIG. 7B). As shown in this figurethe levels of AccA1 and/or AccA2 were not modified by presence of Th.However, cell free-extracts of M94 do contain a slightly higher amountof the 65 kDa protein compared to M86.

[0099] ACCase and PCCase activities were assayed in cell-free extractsof M94 and M86. The levels of both enzyme activities were similar incell-free extracts prepared from cultures of M86 grown in the presenceor in the absence of Th (Table 3). Cell-free extracts prepared frominduced cultures of M94 show instead a remarkable increase in bothACCase (11.5 fold) and PCCase (3.5 fold) activities, compared with thelevels found in non-induced cultures of the same strain or in M86.Moreover, if the enzyme levels found in the wild type strain M145(Rodríguez and Gramajo, 1999) are compared with those found for M94, theincrease in ACCase and PCCase levels were still 4- and 2-fold,respectively (Table 3). These results indicate that by overproducingonly two (β and ε) of the three sub-units that form the acyl-CoAcarboxylase of S. coelicolor we can increase significantly the levels ofthis enzyme activity.

EXAMPLE 8 Influence of the Acyl-CoA Carboxylase Levels in thePhysiological Properties of M94

[0100] Growth curves (FIG. 8A) were determined for the conditionalmutant M94 and for M86 by inoculating a spore suspension in YEME mediumsupplemented with 10 μg of Am, with or without the addition of 5 μg ofTh. For M145, YEME medium without the addition of any antibiotic wasused. M94 supplemented with the inducer (Th) showed a growth rate duringexponential phase very similar to M145, judged from the slope of thecurves. However, the initiation of growth for M94 seems to occur soonerthan in M145, reaching the stationary phase earlier than the wild typestrain. When the cultures were not supplemented with Th, M94 grewconsiderably slower, reaching stationary phase several hours latter thanin the presence of Th. Also, the final OD reached by M94 in the presenceof Th and by M145 were very similar (OD₄₅₀=3) after 60 h of growth.Cultures of M86 grew very slowly compared with M94 and M145,independently of the presence or not of Th. However, these cultureslevelled off at the final OD reached by M145 and M94 after 50 h ofgrowth.

[0101] Actinorhodin and undecylprodigiosin were also quantitatedthroughout growth. Table 4 shows that antibiotic production was onlydetected in cultures of M94 grown in the presence of 1 or 5 μg of Th. Noantibiotic production was observed in cultures of M145 or M94 withoutTh, at least until after 60 h of growth. No antibiotic production wasdetected in M86.

[0102] To determine the effect of Th induction in M86 and M94, 1 μg ofthe antibiotic was spotted to a confluent lawn of these strains in R2and R5 medium supplemented with 10 μg of Am. A striking stimulatoryeffect in both sporulation and antibiotic production was observed in M94after 48 h. No stimulation of growth or antibiotic production wasobserved in M86.

[0103]FIG. 8B shows the stimulatory effect on actinorhodin production inM94 compared to M145 in cultures grown in the presence of 5 μg of Th.

EXAMPLE 9 Co-Expression of AccA, AccB and AccE in S. coelicolor

[0104] The NdeI-XbaI fragment of pTR154 (FIG. 14) is introduced intopIJ8600 and then transformed into S. coelicolor M145 (FIG. 14).Transformants are selected with apramycin and thiostrepton.Overexpression of the three components accA2, accB and accE results inincreased. ACCase activity and antibiotic production compared to thewild type M145 strain.

DISCUSSION

[0105] The use of pccB (Rodríguez and Gramajo, 1999) as an heterologousprobe, allowed the successful isolation of a chromosomal DNA fragmentcontaining accB, a gene encoding for a putative new carboxyl transferaseof S. coelicolor. This predicted function was based on the highpercentage of identity that AccB showed not only to the S. coelicolorPccB, but to several others biochemical and/or genetically characterizedcarboxyl transferases reported for actinomycetes, such as the PccB ofSac. erythraea (Donadio, et al., 1996) and to a less extent to the AccD5of M. tuberculosis (Cole, et al., 1998) and PccB of M. leprae (Doukhan,1995). An interesting finding from the analysis of the cloned sequencewas the presence of a very small ORF, named accE, immediately downstreamof accB.

[0106] The successful expression of accB, accE and the BC-BCCP-(biotincarboxylase- and biotin carboxylase carrier protein-)encoding genesaccA1 and accA2 in E. coli allowed in vitro studies to be performed inorder to understand the role of the corresponding encoded proteins ascomponents of a previously uncharacterized acyl-CoA carboxylase. Thereconstitution, by mixing cell-free extracts of E. coli containing AccBand AccA1 (or AccA2), of an active enzyme with the ability tocarboxylate either acetyl- or propionyl-CoA clearly established thatAccB was the carboxyl transferase component of an acyl-CoA carboxylasecomplex. Interestingly, the small polypeptide, AccE, also showed to playan important role in the reconstitution of a fully active enzyme complex(Table 2). It remains to be elucidated whether this protein plays a roleas an allosteric regulator of the enzyme or whether it is a structuralcomponent of the complex. Thus, our results represent the firstcharacterization, at both the genetic and biochemical levels, of aprokaryotic acyl-CoA carboxylase.

[0107] All the acyl-CoA carboxylases studied so far contain the threefunctional domains in two individual polypeptides (for a review seeBrownsey et al., 1997 ), and none of the purified complexes have shownthe presence of a small component equivalent to AccE. Therefore, thismight be a distinctive feature for Streptomyces sp. In addition, no AccEhomologues have been found in any of the bacteria genomes sequenced sofar, an observation that could also support this hypothesis.

[0108] Malonyl-CoA is an essential component of all living organisms,since it is the main elongation unit for fatty acid biosynthesis(Brownsey et al., 1997). This primary metabolite is synthesised in mostspecies through the carboxylation of acetyl-CoA by an ACCase (Bloch andVance, 1977). If this was also the case for S. coelicolor and, if AccBwas the component of an essential acyl-CoA carboxylase, mutation of thisgene should be lethal for the micro-organism. Replacement of thewild-type accB for the Hyg^(R) mutant allele prove to be unsuccessful,and it only occurred when a second copy of the accBE genes was presentin the chromosome (FIG. 2B).

[0109] These experiments clearly indicated that at least accB wasessential for S. coelicolor viability. The fact that both AccA2(Rodríguez and Gramajo, 1999) and AccB have proved to be essential,along with the fact that acyl-CoA carboxylase reconstituted in vitrowith these two sub-units has the ability to recognise either acetyl- orpropionyl-CoA as substrates, strongly suggests that AccA2 and AccB arethe α and β components of an essential acyl-CoA carboxylase, whose mainphysiological role should be the biosynthesis of malonyl-CoA. Thetranscriptional levels of accB and accA2 throughout growth (FIGS. 3A and4A) also support this interpretation, since both genes are principallytranscribed during exponential and transition phase. Moreover, ACCaseand PCCase activities also showed the highest and constant levels ofactivities during exponential and transition phase while in stationaryphase the activities were low but readily measurable.

[0110] In S. coelicolor, besides the obvious need for malonyl-CoAbiosynthesis during vegetative growth, there is also a requirement forthis metabolite during transition and stationary phase, since at leasttwo secondary metabolites (undecylprodigiosin and actinorhodin) aresynthesised during these growth-phases and they both require malonyl-CoAfor their biosynthesis. Hence, if the ACCase is the only enzyme thatsynthesises malonyl-CoA in this bacterium, its presence will be alsorequired during the idiophase.

[0111] According to the proposed composition of this enzyme complex andbased on the transcriptional studies, we propose that the low level ofexpression of accA2 and accBE during stationary phase is sufficient toproduce enough of the α and β components for an active acyl-CoAcarboxylase. From the observation that accA1 mRNA peaks duringtransition phase, we propose that enough AccA1 might be present in thecytoplasm to compete with AccA2 as the main α sub-unit of this enzymecomplex in the stationary phase. However, no difference in antibioticproduction has been found between M145 and the isogenic accA1 mutant MA4(Rodríguez and Gramajo, 1999).

[0112] We have clearly demonstrated the ability of S. coelicolor toefficiently utilize malonate as a sole carbon and energy source. Aputative pathway for the utilization of this substrate could involve theR. trifolii MatC and MatB homologues which are found in the genome of S.coelicolor. The biochemical characterization of MatB in R. trifoliidemonstrated that this protein is a malonyl-CoA synthetase, whichcatalyzes the formation of malonyl-CoA directly from malonate and CoA.MatC, instead, has not been characterized biochemically but computeranalysis indicate that it is a transmembrane protein that could functionas a dicarboxylate (malonate for example) carrier (An and Kim, 1998). Ifthese enzymes were part of the pathway that allows S. coelicolor toutilize malonate as a sole carbon source, one could also presume thatthe malonyl-CoA synthesized by MatB should fulfill the malonyl-CoArequirements of the micro-organism. However, we could not show thatunder these conditions the essential acyl-CoA carboxylase becomesdispensable.

[0113] Interestingly, the addition of 0.4% malonate to SFM andglucose-MM media produced a clear stimulation of actinorhodin production(data not shown). From this we propose that higher levels of malonyl-CoAwere probably available under this growth conditions. From this, and theobservation that even the limited levels of the ACCase activity foundduring the stationary phase of growth of this bacterium are sufficientto allowed regular levels of antibiotic production, the inventorspropose that increasing the expression of the ACCase components willprobably lead to an improved production of antibiotics.

EXPERIMENTAL PROCEDURES Bacterial Strains, Cultures and TransformationConditions

[0114]S. coelicolor A3 (2) strain M145 (SCP1⁻ SCP2⁻) was manipulated asdescribed by Hopwood et al. (1985). The strain was grown on various agarmedia—SFM (Rodríguez and Gramajo, 1999), R2 and R5—or in 50 ml SMM orYEME liquid media (Hopwood et al (1985) supra). Escherichia coli strainDH5α (Hanahan 1983) was used for routine subcloning and was transformedaccording to Sambrook et al. (1989). Transformants were selected onmedia supplemented with the appropiate antibiotics : ampicillin (Ap) 100μg/ml; apramaycin (Am) 100 μg/ml; chloramphenicol (Cm) 25 μg/ml orkanamycin (Km) 30 μg/ml. Strain BL21(DE3) is an E. coli B strain [F⁻ompT (r_(B) ⁻ m_(B) ⁻) (DE3)] lysogenized with 1DE3, a prophage thatexpresses the T7 RNA polymerase downstream of the IPTG-inducible lacUV5promoter (Studier & Moffat, 1986). ET12567/pUZ8002 (MacNeil et al(1992)/Paget et al (1999)) was used for E. coli-S. coelicolorconjugation experiments (Bierman, 1992). For selection of Streptomycestransformants and exconjugants, media were overlayed with thiostrepton(Th) (300 μg per plate), hygromycin (Hyg) (1 mg per plate) or apramycin(Am) (1 mg per plate). Strains and recombinant plasmids are listed inTable 1.

Growth Conditions, Protein Expression and Preparation of Cell-FreeExtracts

[0115]S. coelicolor M145 was grown at 30° C. in shake flasks in YEMEmedium for 24-48 h. When necessary, 10 mg Am ml⁻¹ or 5 mg Th ml⁻¹ wereadded to the medium. Mycelia were harvested by centrifugation at 5000×gfor 10 min at 4° C., washed in 100 mM potassium phosphate buffer pH 8containing 0.1 mM DTT, 1 mM EDTA, 1 mM PMSF and 10% glycerol (buffer A)and resuspended in 1 ml of the same buffer. The cells were disrupted bysonic treatment (4 or 5 s bursts) using a VibraCell Ultrasonic Processor(Sonics & Materials, Inc.).

[0116] Cell debris was removed by centrifugation and the supernatantused as cell-free extract. For the expression of heterologous proteins,E. coli strain BL21(DE3) harbouring the appropriate plasmids were grownat 37° C. in shake flasks in LB medium in the presence of 25 μg Cm ml⁻¹or 100 μg Ap ml⁻¹ for plasmid maintenance. For the expression ofbiotinylated proteins, 10 μM d-biotin was supplemented to the medium.Overnight cultures were diluted 1:10 in fresh medium and grown to A₆₀₀0.4-0.5 before the addition of IPTG to a final concentration of 0.1 mM.Induction was allowed to proceed for 4 h. The cells were then harvested,washed and resuspended in 1 ml buffer A. Cell-free extracts wereprepared as described above.

Protein Methods

[0117] Cell-free extracts were analysed by denaturing (SDS)-PAGE(Laemmli, 1970) using the Bio Rad mini-gel apparatus. The finalacrylamide monomer concentration was 12% (w/v) for the separating geland 5% for the stacking gel. Coomassie brilliant blue was used to stainprotein bands. The biotinylated proteins were detected by a modificationof the Western blotting procedure described by Nikolau et al. (1985).After electrophoretic separation, proteins were electro-blotted ontonitrocellulose membranes (Bio-Rad) and probed with alkalinephosphatase-streptavidin conjugate (Bio-Rad) diluted 1:10000. Proteincontent was determined by the method of Bradford (1976) with BSA asstandard.

In Vitro Reconstitution and Assay of the Acyl-CoA Carboxylase Complex

[0118] In vitro reconstitution of the enzyme complex was carried out bymixing 100 μg of each of the cell-free extracts shown in Table 2 in afinal volume of 300 μl. When AccE was not included in the incubationmix, 100 μg of BSA were added instead. The mixes were incubated for 1 hat 4° C. and 100 μg of each used for enzyme assay.

[0119] ACCase and PCCase activities in cell-free extracts were measuredfollowing the incorporation of H¹⁴CO₃ ⁻ into acid non-volatile material(Huanaiti & Kolattukudy, 1982; Bramwell et al., 1996). The reactionmixture contained 100 mM potassium phosphate pH 8.0, 300 μg BSA, 3 mMATP, 5 mM MgCl₂, 50 mM NaH¹⁴CO₃ [specific activity 200 μCi mmol⁻¹ (740kBq mmol⁻¹)], 1 mM substrate (acetyl-CoA or propionyl-CoA) and 100 μgcell-free protein extract in a total reaction volume of 100 μl. Thereaction was initiated by the addition of NaH¹⁴CO₃, allowed to proceedat 30° C. for 15 min and stopped with 200 μl 6 M HCl. The contents ofthe tubes were then evaporated to dryness at 95° C. The residue wasresuspended in 100 μl water, 1 ml of Optiphase liquid scintillation(Wallac Oy) was added and ¹⁴C radioactivity determined in a Beckmanscintillation liquid counter. Non-specific CO₂ fixation by crudeextracts was assayed in the absence of substrate. One unit of enzymeactivity catalysed the incorporation of 1 μmol ¹⁴C into acid-stableproducts per min.

DNA Manipulations

[0120] Isolation of chromosomal and plasmid DNA, restriction enzymedigestion and agarose gel electrophoresis were carried out byconventional methods (Sambrook et al., 1989; Hopwood et al., 1985).Southern analyses were performed by using P-labelled probes made byrandom oligonucleotide priming (Prime-a-gene kit; Promega).

Gene Cloning and Plasmid Construction

[0121] The synthetic oligonucleotides TC1, 5′-CAGAATTCAAGCAGCACGCCAAGGGCAAG, and TC2, 5′-CAGAATTCGATGCCGTCGTGCTCCTGGTC, were used to amplify aninternal fragment of the S. coelicolor pccB gene. The reaction mixturecontained 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1 mM MgCl₂, 6% glycerol, 25μM of each of the four dNTPs, 2.5 U Taq DNApolymerase, 20 pmol of eachprimer and 50 ng of S. coelicolor chromosomal DNA in a final volume of100 μl. Samples were subjected to 30 cycles of denaturation (95° C., 30s), annealing (65° C., 30 s) and extension (72° C., 1 min). A 1 kb PCRfragment was used as a ³²P-labelled probe to screen a size-enrichedlibrary. A 2.7 kb BamHI fragment containing an incomplete accB gene wascloned in BamHI-cleaved pBluescript SK(+), yielding pTR62.

[0122] The synthetic oligonucleotide TC16(5′-TATTCTAGACATATGACCGTTTTGGATGAGG, used to introduce an NdeI site atthe translational start codon of the S. coelicolor accB gene) and TC17(5′-ACCTCTAGACAACGCTCGTGGACC, used to introduce an XbaI site in the accBcoding sequence) were used to amplify an internal fragment of S.coelicolor accB gene, having the sequence shown in FIG. 10. The reactionmixture was the same as the one indicated above. Samples were subjectedto 30 or 35 cycles of denaturation (95° C., 30 s), annealing (65° C., 30s) and extension (72° C., 1 min). The 1 kb PCR product was digested withNdeI and XbaI (these sites were introduced in the 5′ ends of the oligosTC16 and TC17 and are shown in bold in FIG. 10) and cloned inXbaI-cleaved pBluescript SK(+) in E. coli DH5α, yielding pTR82. Thisplasmid was digested with BstEII and SacI, ligated with a BstEII-SacIfragment cleaved from pRM08 and introduced by transformation into E.coli DH5α, yielding pTR87.

[0123] An NdeI-XbaI fragment from the plasmid pTR82 was cloned inNdeI-XbaI-cleaved pIJ8600 (Sun et al (1999)), yielding pTR93. In orderto place the chromosomal copy of accBE operon under the tipA promoter weremoved from pTR93 a HindIII fragment containing the int gene and att ofΦC31, yielding pTR94. Plasmid pTR94 was transformed into strainET12567/pUZ8002 and transferred by conjugation to S. coelicolor M145(Hopwood et al (1985)).

[0124] A NdeI-SacI fragment from the plasmid pTR87 was cloned inNdeI-SacI-cleaved pET22b(+) (Novagen) (pTR88), thus placing the accBEoperon under the control of the powerful T7 promoter andribosome-binding sequences. The synthetic oligonucleotides NaccE,5′-TTATCTAGACATATGTCCCCTGCCGAC, used to introduce an NdeI site at thetranslational start codon of the S. coelicolor accE gene, and CaccE,5′-ATGAATTCTATGCATCGGGTCAGCGCCAGCTG, were used to amplify the accE geneof S. coelicolor. The reaction mixture was the same as the one indicatedabove. Samples were subjected to 35 cycles of denaturation (95° C., 30s), annealing (65° C., 30 s) and extension (72° C., 30 s). The PCRproduct was cloned using pGEM-T easy vector (Promega) in E. coli DH5α,yielding pTR106. A NdeI-EcoRI fragment from the plasmid pTR106 wascloned in NdeI-EcoRI-cleaved pET22(b) (Novagen) yielding the plasmidpTR107, thus placing the accE gene under the control of the powerful T7promoter and ribosome-binding sequences.

[0125] Plasmid pIJ8600 was digested with BglII and EcoRI and thefragment containing oriT RK2, ori pUC18, attP site, int ΦC31 andaac(3)IV (Am^(R) cassette) genes was ligated with a linker containingthe following enzymes (Mike Butler personal comunication): BglII, AseI,EcoRI, BglII, NdeI, KpnI, XbaI, PstI, HindIII, BamHI, SstI, NotI andEcoRI, yielding pTR141. A 4.0 kb KpnI fragment containing the completeaccBE operon from pRM08 was cloned into KpnI-cleaved pTR141, yieldingpTR149.

[0126] For an efficient over-expression in S. coelicolor of the threecomponents of the acyl-CoA carboxylase complex of this micro-organism,we carried out the construction of pTR156 through the following steps.First we did a PCR amplification of the chromosomal accBE operon usingthe oligo TC16 (5′-TATTCTAGACATATGACCGTTTTGGATGAGG 3′), to introduce aNdeI site at the translation start codon of accB, and the oligo C-accE(5′ATG AAT TCT ATG CAT CGG GTC AGC GCC AGC 3′) to introduce a NsiIrestriction site at the 3′ end of accE. The amplified DNA, was thencloned into pGEM-T (Promega), to give pTR99. To introduce a NsiI siteupstream of the RBS of accA2 we amplified this gene using the oligoN-accA2 (5′ ATG AAT TCA TGC ATG AGG GAG CCT CAA TCG 3′), for the 5′ endand the oligo C-accA2 (5′ AGA TCT AGA TCA GTC CTT GAT CTC GC 3′)containing a XbaI and a EcoRI site, for the 3′ end of the gene. Theamplified DNA was cloned in pGEM-T to give pTR112. The NdeI-NsiI DNAfragment from pTR99 and the NsiI-EcoRI fragment isolated from pTR112were finally cloned into pET22(b) (Stratagene), previously digested withNdeI and EcoRI, to yield pTR154. In order to introduce these genes in S.coelicolor we sub-cloned the NdeI-XbaI fragment, containing accBE andaccA2, from pTR154 to pIJ8600 digested with the same enzymes to givepTR156. See FIG. 14 for plasmid constructions.

Nucleotide Sequencing

[0127] The sequence of the SphI original fragment was performed fromplasmids DNA constructed by subcloning ApaI DNA fragments from pRM08into pSKBluescribe SK(+). Synthetic oligonucleotides were used tocomplete the sequence. The nucleotide sequence of the accBE region wasdetermined by dideoxy sequencing (Sanger et al., 1977) using the PromegaTaqTrack sequencing kit and double-stranded DNA templates. The completesequence of the 1C2 cosmid, that includes the SphI fragment harbouringaccBE, is available from the S. coelicolor genome sequencing project.

S1 Nuclease Mapping

[0128] For each S1 nuclease reaction, 30 μg of RNA were hybridized inNaTCA buffer (Murray, 1986); solid NaTCA (Aldrich) was dissolved to 3Min 50 mM PIPES (pH 7.0), 5 mM EDTA, to about 0.002 pmol (approximately10⁴ cpm) of the following probes. For accA2 the syntheticoligonucleotide 5′-GCTTTGAGGACCTTGGCGATG (accA2down), corresponding tothe sequence within the coding region of accA2, was uniquely labelled atthe 5′ end of the oligonucleotide with [³²P]-ATP using T4 polynucleotidekinase. The labelled oligo was then used in the PCR reaction with theunlabelled oligonucleotide (accA2up) 5′-GAAGTACAGGCCGAAGACCAC, whichcorresponds to a region upstream of the accA2 promoter region, togenerate a 766 bp probe. For accA1 the synthetic oligonucleotide(accA1down) 5′-GCGATTTCGCCACGATTGGCG, corresponding to the region withinthe coding region of accA1, was uniquely labelled with [³²P]-ATP usingT4 polynucleotide kinase at the 5′ end of the oligonucleotide. TheaccA1down oligo was later used in the PCR reaction with the unlabelledoligonucleotide (accA1up) 5′-CCGATATCAGCCCCTGATGAC, which corresponds toa region upstream of the accA1 promoter to generate a 563 bp probe. ForaccB the synthetic oligonucleotide (accBdown) 5′-CGTCAGCTTGCCCTTGGCGTG,corresponding to the region within the coding region of accB, wasuniquely labelled with [³²P]-ATP using T4 polynucleotide kinase at the5′ end of the oligonucleotide. accBdown was then used in the PCRreaction with the unlabelled oligonucleotide (accBup)5′-CTACGCTCCGGGTGAGCGAAC, which corresponds to a region upstream of theaccB promoter, to generate a 483 bp probe. For accBE the syntheticoligonucleotide (accBEdown) 5′-GGAGGGCCGTGATGGCGGCGACTTCCTCGGG,corresponding to the region within the coding region of accE wasuniquely labelled with [³²P]-ATP using T4 polynucleotide kinase at the5′ end of the oligonucleotide. The accBEdown oligo was then used in thePCR reaction with the unlabelled oligonucleotide (accBEup)5′-GAGGAACTGGTACGCGCGGGCG(GTACAAGCAAGCT), which corresponds to a regionwithin the coding region of accB (bracketed oligonucleotides are a tailadded to the probe to differentiate probe reannealing from fullyprotected DNA-RNA complexes), to generate a 563 bp probe. Subsequentsteps were as described by Strauch et al. (1991).

Determination of Actinorhodin

[0129] 1 ml of whole broth was mixed with 0.5 ml of 3N KOH to give afinal concentration of 1N KOH. The solutions were mixed vigorously andcentrifuge at 4000×g for 5 minutes. The supernatant was collected andmeasured at A_(640 nm). Actinorhodin concentration was calculated usingthe molar extinction coefficient (in 1 N KOH) at 640 nm of 25.320(Bystrykh et al., 1996).

Determination of Undecylprodigiosin

[0130] This was carried out according to the procedure of Hobbs et al.(1990).

REFERENCES

[0131] An, J. H. et al. (1998) Eur J Biochem 257: 395-402.

[0132] Behal, V. et al. (1977) Phytochemistry 16: 347-350.

[0133] Bierman, M. et al. (1992) Gene 116: 43-49.

[0134] Bloch, K. and Vance, D. (1977) Annu Rev Biochem 46: 263-298.

[0135] Bradford, M. (1976) Anal Biochem 72:248.

[0136] Bramwell, H. et al. (1996) Microbiol 142: 649-655.

[0137] Bramwell, H. et al. (1993) Biochem J 293:131-136.

[0138] Brownsey, R. W. et al. (1997) Biochem Soc Trans 25: 1232-1238.

[0139] Buttner, M. J. et al. (1990) J Bacteriol 172: 3367-3378.

[0140] L. V. Bystrykh, M. A. et al. (1996). J Bacteriol. 187: 2238-2344.

[0141] Cole, S. T. et al. (1998) Nature 393: 537-544.

[0142] Donadio, S. et al. (1996) Mol Microbiol 19: 977-984.

[0143] Doukhan, L. et al. (1995) Gene 165: 67-70

[0144] Erfle, J. D. (1973) Biocim Biophys Acta 316: 143-155.

[0145] Gramajo, H. C. et al. (1993) Molecular Microbiology 7: 837-845.

[0146] Hanahan (1983) J. Mol. Biol. 166: 557-580.

[0147] Harwood, J. L. (1988) Annu Rev Plant Physiol Plant Mol Biol 39:101-138.

[0148] Hasslacher, M. et al. (1993) J. Biol. Chem. 268: 10946-10952.

[0149] Henrikson, K. P. and Allen, S. H. G. (1979) J Biol Chem254:5888-5891.

[0150] Hobbs, G. et al. (1990) J Gen Microbiol 136:2291-2296.

[0151] Hopwood, D. A. et al. (1985) Genetic manipulation ofStreptomyces: A laboratory manual. John Innes Foundation, Norwich.

[0152] Hopwood, D. A. and Sherman, D. H. (1990) Ann Rev Genet 24:37-66.

[0153] Hunaiti, A. R. and Kolattukudy, P. E. (1982) Arch Biochem Biophys216:362-371.

[0154] Laakel, M. et al. (1994) Microbiol 140: 1451-1456.

[0155] Laemmli, U. K. (1970) Nature 227:680-685.

[0156] Li, S. J. and Cronan. J. E., Jr. (1993) J Bacteriol 175: 332-340.

[0157] MacNeil et al. (1992) Gene 115: 119-125

[0158] Murakami, T. et al. (1989) J Bacteriol 171: 1459-1466.

[0159] Murray (1986) Anal Biochem 158(1): 165-170.

[0160] Nikolau, B. J. et al. (1981) Arch Biochem Biophys 211: 605-612.

[0161] Paget et al. (1999) Mol. Microbiol. 3: 97-107

[0162] Perez, C. A. et al. (1998) Microbiology 144: 895-903.

[0163] Pizer, E. S. et al. (1996) Cancer Res. 56: 2745-7.

[0164] Polakis, S. et al. (1972) J Biol Chem 247: 1335-1337.

[0165] Redenbach, M. et al. (1996) Mol Microbiol 21: 77-95.

[0166] Rodríguez, E. and Gramajo, H. (1999) Microbiology 145: 3109-3119.

[0167] Saggerson, E. D. et al. (1992) Adv Enzyme Regul 32: 285-306.

[0168] Sambrook, J. et al. (1989) Molecular Cloning: a LaboratoryManual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

[0169] Sanger, F. et al. (1977) Proc Natl Acad Sci USA 74: 5463-5467.

[0170] Strauch, E. et al. (1991) Mol Microbiol 5: 289-298.

[0171] Strohl, W. R. (1992) Nucleic Acids Res. 20: 961-974.

[0172] Studier and Moffatt 1986 J Mol Biol 189(1): 113-30.

[0173] Sun et al. (1999) Microbiology 145: 2221-2227

[0174] Westrich, L. et al. (1999) FEMS Microbiol. Lett. 170: 381-387.

[0175] All the above references are hereby incorporated by reference intheir entirety, individually and for all purposes. TABLE 1 Strains andplasmids used. Strain/plasmids Description Reference/source S.coelicolor M145 Parental strain SCP 1⁻ SGP2⁻ Hopwood et al. (1985) T124M145 (accB:pTR124), Th^(R), Hyg^(R) This work T149 T124 containingpTR149 integrated in the att site This work of φC31, Th^(R), Hyg^(R),Am^(R) T149A T149 with the wilde type accB copy of the This workchromosome replaced by the accB::hyg mutant allele, Hyg^(R), Am^(R) E.coli DH5α F⁻ ΔlacU169 (φ80lacZΔM15) endA1 recA1 Hanahan (1983) hsdR17deoR supE44 thi-I λ⁻gyrA96 relA1 BL2Iλ(DE3) F′ ompTr_(B) ⁻m_(B) ⁻(DE3)Studier & Moffatt (1986) ET 12567 supE44 hrdS20 (r⁻ _(B)m⁻ _(B)) ara-14pro A2 lacY galK2 MacNeil et al. (1992) rpsL20 xyl-5 mtl-1 dam⁻ dcm⁻hsdM Cm^(R) RG7 DH5α carrying pCL1 and pBA11 plasmids Rodriguez &Gramajo (1999) Plasmids pBluescript SK(+) Phagemid vector (Ap^(R) lacZ′)Stratagene pGEM-T Easy For cloning PCR products Promega pIJ2925 pUC18derivative (Ap^(R) lacZ′) Janssen & Bibb (1993) pSET151 For the conjugaltransfer of DNA from E. coli to Bierman et al. (1992) Streptomyces spp.(Ap^(R) Th^(R) lacZ′) pET22b(+) Phagemid vector (Ap^(R) lacZ′) forexpression of Novagen recombinant proteins under control of strong T7transcription and translation signals pUZ8002 RK2 derivative withdefective oriT (Km^(R)) Paget et al. (1999) pIJ8600 For the conjugaltransfer of DNA from E. coli to Sun et al. (1999) Streptomyces spp. andfor expression of recombinant proteins under tipA promoter pBA11 Vectorcontaining E. coli birA gene Barker & Campbell (1981) pCL1 pSK(+) with aEcoRI-KpnI insert carrying accA1 Rodriguez & Gramajo (1999) pMR08 pSK(+)with a SstI insert carrying accBE This work pTR88 pET22b(+) with accBEunder control of strong T7 This work transcription and translationsignals pTR90 pET22b(+) with accB under control of strong T7 This worktranscription and translation signals pTR107 pET22b(+) with accE undercontrol of strong T7 This work transcription and translation signalspTR124 pSET151 with a hyg (Hyg^(R)) gene inserted in the This work accEcoding region pTR141 pIJ8600 derivative carrying oriT RK2, ori pUC18,This work attP site, int φC31 and aac(3)IV (Am^(R)) pTR149 pTR141 with aKpnI insert carrying accBE This work

[0176] TABLE 2 Heterologous expresion of acyl-CoA carboxylase componentsin cell-free extracts of E. coli and in vitro reconstitution of enzymeactivity Cell-free extracts Strain Proteins ACCase PCCase E. coli*induced by IPTG [mU (mg protein)⁻¹]⁺ [mU (mg protein)⁻¹]⁺ RG7 AccA1 +BirA ND ND RG8 AccB, AccE ND ND RG9 AccB ND ND RG10 AccE ND NDRG7:RG8^(&) AccA1 + BirA:AccB, AccE 2.35 ± 0.06 3.10 ± 0.07 RG7:RG9^(&)AccA1 + BirA;AccB 0.32 ± 0.05 0.50 ± 0.05 RG7:RG9:RG10^(&) AccA1 +BirA:AccB;AccE 1.38 ± 0.05 1.77 ± 0.06

[0177] TABLE 3 ACCase and PCCase activities in M145, M86 and M94 StrainInduction Activity S. with ACCase PCCase coelicolor Thiostrepton [mU (mgprotein)^(−1]*) [mU (mg protein)^(−1]*)  M145 − 1.12 ± 0.03  2.2 ± 0.03M86 − 0.43 ± 0.03 1.45 ± 0.06 M86 + 0.33 ± 0.03 0.95 ± 0.06 M94 − 0.40 ±0.03 1.57 ± 0.03 M94 + 4.61 ± 0.03 (11.5) 5.41 ± 0.03 (3.5)

[0178] TABLE 4 Production of actinorhodin and undecylprodigiosin in YEMEmedium by M145 and M94. M145 M94 [M94 + 1 μg Th]* [M94 + 5 μg Th]* Time(h) Act Red Act Red Act (M) Red (M) Act (M) Red (M) 40 — — — — 3 × 10⁻⁷10 × 10⁻⁷ 11 × 10⁻⁷ 28 × 10⁻⁷ 60 — — — — 9 × 10⁻⁷ 12 × 10⁻⁷ 26 × 10⁻⁷ 78× 10⁻⁷

[0179]

1 32 1 29 DNA Artificial Synthetic oligonucleotide 1 cagaattcaagcagcacgcc aagggcaag 29 2 29 DNA Artificial Synthetic oligonucleotide 2cagaattcga tgccgtcgtg ctcctggtc 29 3 31 DNA Artificial Syntheticoligonucleotide 3 tattctagac atatgaccgt tttggatgag g 31 4 24 DNAArtificial Synthetic oligonucleotide 4 acctctagac aacgctcgtg gacc 24 527 DNA Artificial Synthetic oligonucleotide 5 ttatctagac atatgtcccctgccgac 27 6 32 DNA Artificial Synthetic oligonucleotide 6 atgaattctatgcatcgggt cagcgccagc tg 32 7 30 DNA Artificial Syntheticoligonucleotide 7 atgaattcta tgcatcgggt cagcgccagc 30 8 30 DNAArtificial Synthetic oligonucleotide 8 atgaattcat gcatgaggga gcctcaatcg30 9 26 DNA Artificial Synthetic oligonucleotide 9 agatctagat cagtccttgatctcgc 26 10 21 DNA Artificial Synthetic oligonucleotide 10 gctttgaggaccttggcgat g 21 11 21 DNA Artificial Synthetic oligonucleotide 11gaagtacagg ccgaagacca c 21 12 21 DNA Artificial Syntheticoligonucleotide 12 gcgatttcgc cacgattggc g 21 13 21 DNA ArtificialSynthetic oligonucleotide 13 ccgatatcag cccctgatga c 21 14 21 DNAArtificial Synthetic oligonucleotide 14 cgtcagcttg cccttggcgt g 21 15 21DNA Artificial Synthetic oligonucleotide 15 ctacgctccg ggtgagcgaa c 2116 31 DNA Artificial Synthetic oligonucleotide 16 ggagggccgt gatggcggcgacttcctcgg g 31 17 35 DNA Artificial Synthetic oligonucleotide 17gaggaactgg tacgcgcggg cggtacaagc aagct 35 18 120 DNA Streptomycescoelicolor 18 aaacgggccg gagactgtac ggagtcgacg gctcgcaatc cttgctcggcttcgtagagt 60 cgctacatga ccgttttgga tgaggcgccg ggcgagccga cggacgcgcgcgggcgggtg 120 19 120 DNA Streptomyces coelicolor 19 cacccgcccgcgcgcgtccg tcggctcgcc cggcgcctca tccaaaacgg tcatgtagcg 60 actctacgaagccgagcaag gattgcgagc cgtcgactcc gtacagtctc cggcccgttt 120 20 18 PRTStreptomyces coelicolor 20 Met Thr Val Leu Asp Glu Ala Pro Gly Glu ProThr Asp Ala Arg Gly 1 5 10 15 Arg Val 21 240 DNA Streptomyces coelicolor21 cagccatgtc tcacgagccg gtaaccccgg ggcaaccgcg aaccggacgg ccccttccgg 60cactggtcct gctccgcccg gggggcgggt gattaccgcg ggatggacgg gcggctgaat 120accggccgat acgttctgcg catgactgtt ccgaacaggg cggcgtgaat tccaaccgtt 180ggccgtcggc gagccccgat cagtaatcga gtgagtgagg agaatcttcg tgcgcaaggt 240 22240 DNA Streptomyces coelicolor 22 ccacaaagaa accgcgtggc ccgcagcacgcccttacaga gaccttgacc acacaggagg 60 gctagggttt cccccaggag tcctgcgtaccgcggtacta caagggcttt cgggggtcga 120 gcgagcctcg aatcacgctc cgtgtgggcaagctcaccat tggggacggg tcgaagtgcc 180 gtgtcggcag tccctaaact cggcttgtttcaaggaggga gcctcaatcg tgcgcaaggt 240 23 4 PRT Streptomyces coelicolor 23Val Arg Val Lys 1 24 968 DNA Streptomyces coelicolor 24 tattctagacatatgaccgt tttggatgag gcgccgggcg agccgacgga cgcgcgcggg 60 cgggtggccgagctgcacgg gatccgtgca gcggcgctcg ccgggccgag tgagaaggcg 120 acggcggcgcagcacgccaa gggcaagctg acggcacgtg agcgcatcga gctgctcctg 180 gaccccggctccttccgcga ggtcgagcag ctgcgccggc accgggcgac cgggttcggc 240 ctggaggccaagaagccgta caccgacggt gtcatcaccg gctggggcac ggtcgagggc 300 cgcacggtcttcgtctacgc ccacgacttc cggatcttcg gcggcgcgct gggcgaggcc 360 cacgccacgaagatccacaa gatcatggac atggccatcg cggccggtgc cccgctggtg 420 tcgctgaacgacggcgccgg cgcccgtatc caggagggcg tcagcgcgct cgccgggtac 480 ggcggcatcttccagcgcaa caccaaggcg tccggcgtca tcccgcagat cagcgtgatg 540 ctcggcccctgcgcgggcgg cgcggcctac agccccgccc tcaccgactt cgtcttcatg 600 gtccgcgacacctcgcagat gttcatcacg ggcccggacg tcgtcaaggc ggtcaccggc 660 gaggagatcacgcagaacgg tctgggcggc gccgacgtgc acgccgagac gtccggcgtg 720 tgccacttcgcctacgacga cgaggagacc tgcctcgccg aggtccgcta cctcctctcc 780 ctcctcccgcagaacaaccg ggagaacccg ccccgcgccg agtcctccga ccccgtggac 840 cgccgctcggacaccctcct cgacctggtc ccggcggacg gcaaccgccc gtacgacatg 900 accaaggtcatcgaggaact cgtcgacgag ggcgagtacc tggaggtcca cgagcgttgt 960 ctagaggt 96825 590 PRT Streptomyces coelicolor 25 Val Arg Lys Val Leu Ile Ala AsnArg Gly Glu Ile Ala Val Arg Val 1 5 10 15 Ala Arg Ala Cys Arg Asp AlaGly Ile Ala Ser Val Ala Val Tyr Ala 20 25 30 Asp Pro Asp Arg Asp Ala LeuHis Val Arg Ala Ala Asp Glu Ala Phe 35 40 45 Ala Leu Gly Gly Asp Thr ProAla Thr Ser Tyr Leu Asp Ile Ala Lys 50 55 60 Val Leu Lys Ala Ala Arg GluSer Gly Ala Asp Ala Ile His Pro Gly 65 70 75 80 Tyr Gly Phe Leu Ser GluAsn Ala Glu Phe Ala Gln Ala Val Leu Asp 85 90 95 Ala Gly Leu Ile Trp IleGly Pro Pro Pro His Ala Ile Arg Asp Arg 100 105 110 Gly Glu Lys Val AlaAla Arg His Ile Ala Gln Arg Ala Gly Ala Pro 115 120 125 Leu Val Ala GlyThr Pro Asp Pro Val Ser Gly Ala Asp Glu Val Val 130 135 140 Ala Phe AlaLys Glu His Gly Leu Pro Ile Ala Ile Lys Ala Ala Phe 145 150 155 160 GlyGly Gly Gly Arg Gly Leu Lys Val Ala Arg Thr Leu Glu Glu Val 165 170 175Pro Glu Leu Tyr Asp Ser Ala Val Arg Glu Ala Val Ala Ala Phe Gly 180 185190 Arg Gly Glu Cys Phe Val Glu Arg Tyr Leu Asp Lys Pro Arg His Val 195200 205 Glu Thr Gln Cys Leu Ala Asp Thr His Gly Asn Val Val Val Val Ser210 215 220 Thr Arg Asp Cys Ser Leu Gln Arg Arg His Gln Lys Leu Val GluGlu 225 230 235 240 Ala Pro Ala Pro Phe Leu Ser Glu Ala Gln Thr Glu GlnLeu Tyr Ser 245 250 255 Ser Ser Lys Ala Ile Leu Lys Glu Ala Gly Tyr GlyGly Ala Gly Thr 260 265 270 Val Glu Phe Leu Val Gly Met Asp Gly Thr IlePhe Phe Leu Glu Val 275 280 285 Asn Thr Arg Leu Gln Val Glu His Pro ValThr Glu Glu Val Ala Gly 290 295 300 Ile Asp Leu Val Arg Glu Met Phe ArgIle Ala Asp Gly Glu Glu Leu 305 310 315 320 Gly Tyr Asp Asp Pro Ala LeuArg Gly His Ser Phe Glu Phe Arg Ile 325 330 335 Asn Gly Glu Asp Pro GlyArg Gly Phe Leu Pro Ala Pro Gly Thr Val 340 345 350 Thr Leu Phe Asp AlaPro Thr Gly Pro Gly Val Arg Leu Asp Ala Gly 355 360 365 Val Glu Ser GlySer Val Ile Gly Pro Ala Trp Asp Ser Leu Leu Ala 370 375 380 Lys Leu IleVal Thr Gly Arg Thr Arg Ala Glu Ala Leu Gln Arg Ala 385 390 395 400 AlaArg Ala Leu Asp Glu Phe Thr Val Glu Gly Met Ala Thr Ala Ile 405 410 415Pro Phe His Arg Thr Val Val Arg Asp Pro Ala Phe Ala Pro Glu Leu 420 425430 Thr Gly Ser Thr Asp Pro Phe Thr Val His Thr Arg Trp Ile Glu Thr 435440 445 Glu Phe Val Asn Glu Ile Lys Pro Phe Thr Thr Pro Ala Asp Thr Glu450 455 460 Thr Asp Glu Glu Ser Gly Arg Glu Thr Val Val Val Glu Val GlyGly 465 470 475 480 Lys Arg Leu Glu Val Ser Leu Pro Ser Ser Leu Gly MetSer Leu Ala 485 490 495 Arg Thr Gly Leu Ala Ala Gly Ala Arg Pro Lys ArgArg Ala Ala Lys 500 505 510 Lys Ser Gly Pro Ala Ala Ser Gly Asp Thr LeuAla Ser Pro Met Gln 515 520 525 Gly Thr Ile Val Lys Ile Ala Val Glu GluGly Gln Glu Val Gln Glu 530 535 540 Gly Asp Leu Ile Val Val Leu Glu AlaMet Lys Met Glu Gln Pro Leu 545 550 555 560 Asn Ala His Arg Ser Gly ThrIle Lys Gly Leu Thr Ala Glu Val Gly 565 570 575 Ala Ser Leu Thr Ser GlyAla Ala Ile Cys Glu Ile Lys Asp 580 585 590 26 590 PRT Streptomycescoelicolor 26 Val Arg Lys Val Leu Ile Ala Asn Arg Gly Glu Ile Ala ValArg Val 1 5 10 15 Ala Arg Ala Cys Arg Asp Ala Gly Ile Ala Ser Val AlaVal Tyr Ala 20 25 30 Asp Pro Asp Arg Asp Ala Leu His Val Arg Ala Ala AspGlu Ala Phe 35 40 45 Ala Leu Gly Gly Asp Thr Pro Ala Thr Ser Tyr Leu AspIle Ala Lys 50 55 60 Val Leu Lys Ala Ala Arg Glu Ser Gly Ala Asp Ala IleHis Pro Gly 65 70 75 80 Tyr Gly Phe Leu Ser Glu Asn Ala Asp Phe Ala GlnAla Val Leu Asp 85 90 95 Ala Gly Leu Ile Trp Ile Gly Pro Pro Pro His AlaIle Arg Asp Arg 100 105 110 Gly Glu Lys Val Ala Ala Arg His Ile Ala GlnArg Ala Gly Ala Pro 115 120 125 Leu Val Ala Gly Thr Pro Asp Pro Val SerGly Ala Asp Glu Val Val 130 135 140 Ala Phe Ala Lys Glu His Gly Leu ProIle Ala Ile Lys Ala Ala Phe 145 150 155 160 Gly Gly Gly Gly Arg Gly LeuLys Val Ala Arg Thr Leu Glu Glu Val 165 170 175 Pro Glu Leu Tyr Asp SerAla Val Arg Glu Ala Val Ala Ala Phe Gly 180 185 190 Arg Gly Glu Cys PheVal Glu Arg Tyr Leu Asp Lys Pro Arg His Val 195 200 205 Glu Thr Gln CysLeu Ala Asp Thr His Gly Asn Val Val Val Val Ser 210 215 220 Thr Arg AspCys Ser Leu Gln Arg Arg His Gln Lys Leu Val Glu Glu 225 230 235 240 AlaPro Ala Pro Phe Leu Ser Glu Ala Gln Thr Glu Gln Leu Tyr Ser 245 250 255Ser Ser Lys Ala Ile Leu Lys Glu Ala Gly Tyr Val Gly Ala Gly Thr 260 265270 Val Glu Phe Leu Val Gly Met Asp Gly Thr Ile Ser Phe Leu Glu Val 275280 285 Asn Thr Arg Leu Gln Val Glu His Pro Val Thr Glu Glu Val Ala Gly290 295 300 Ile Asp Leu Val Arg Glu Met Phe Arg Ile Ala Asp Gly Glu GluLeu 305 310 315 320 Gly Tyr Asp Asp Pro Ala Leu Arg Gly His Ser Phe GluPhe Arg Ile 325 330 335 Asn Gly Asp His Pro Gly Arg Gly Phe Leu Pro AlaPro Gly Thr Val 340 345 350 Thr Leu Phe Asp Ala Pro Thr Gly Pro Gly ValArg Leu Asp Ala Gly 355 360 365 Val Glu Ser Gly Ser Val Ile Gly Pro AlaTrp Asp Ser Leu Leu Ala 370 375 380 Lys Leu Ile Val Thr Gly Arg Thr ArgAla Glu Ala Leu Gln Arg Ala 385 390 395 400 Ala Arg Ala Leu Asp Glu PheThr Val Glu Gly Met Ala Thr Ala Ile 405 410 415 Pro Phe His Arg Thr ValVal Arg Asp Pro Ala Phe Ala Pro Glu Leu 420 425 430 Thr Gly Ser Thr AspPro Phe Thr Val His Thr Arg Trp Ile Glu Thr 435 440 445 Glu Phe Val AsnGlu Ile Lys Pro Phe Thr Thr Pro Ala Asp Thr Glu 450 455 460 Thr Asp GluGlu Ser Gly Arg Glu Thr Val Val Val Glu Val Gly Gly 465 470 475 480 LysArg Leu Glu Val Ser Leu Pro Ser Ser Leu Gly Met Ser Leu Ala 485 490 495Arg Thr Gly Leu Ala Ala Gly Ala Arg Pro Lys Arg Arg Ala Ala Lys 500 505510 Lys Ser Gly Pro Ala Ala Ser Gly Asp Thr Leu Ala Ser Pro Met Gln 515520 525 Gly Thr Ile Val Lys Ile Ala Val Glu Glu Gly Gln Glu Val Gln Glu530 535 540 Gly Asp Leu Ile Val Val Leu Glu Ala Met Lys Met Glu Gln ProLeu 545 550 555 560 Asn Ala His Arg Ser Gly Thr Ile Lys Gly Leu Thr AlaGlu Val Gly 565 570 575 Ala Ser Leu Thr Ser Gly Ala Ala Ile Cys Glu IleLys Asp 580 585 590 27 1773 DNA Streptomyces coelicolor 27 gtgcgcaaggtgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60 cgggacgccgggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120 gtccgtgccgctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180 gacatcgccaaggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240 tacggattcctctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300 tggatcggcccgcccccgca cgccatccgc gaccgtggcg aaaaggtcgc cgcccgccac 360 atcgcccagcgggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420 gacgaggtcgtcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480 ggcggcggcgggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540 gactccgccgtccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600 tacctcgacaagccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660 gtcgtcgtctccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720 gcccccgcgccctttctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780 atcctgaaggaggccggcta cggcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840 ggcacgatcttcttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900 gaagtcgccggcatcgactt ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960 ggttacgacgaccccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020 cccggccgcggcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080 cccggcgtccgcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140 tccctcctcgccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200 gcccgcgccctggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260 acggtcgtccgcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320 gtccacacccggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380 gccgacaccgagacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440 aagcgcctggaagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500 gccgccggggcccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560 gacaccctcgcctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggaaggccag 1620 gaagtccaggaaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680 aacgcccacaggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740 tccggcgccgccatctgcga gatcaaggac tga 1773 28 1773 DNA Streptomyces coelicolor 28gtgcgcaagg tgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60cgggacgccg ggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120gtccgtgccg ctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180gacatcgcca aggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240tacggattcc tctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300tggatcggcc cgcccccgca cgccatccgc gaccgtggcg aaaaggtcgc cgcccgccac 360atcgcccagc gggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420gacgaggtcg tcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480ggcggcggcg ggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540gactccgccg tccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600tacctcgaca agccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660gtcgtcgtct ccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720gcccccgcgc cctttctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780atcctgaagg aggccggcta cggcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840ggcacgatct tcttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900gaagtcgccg gcatcgactt ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960ggttacgacg accccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020cccggccgcg gcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080cccggcgtcc gcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140tccctcctcg ccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200gcccgcgccc tggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260acggtcgtcc gcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320gtccacaccc ggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380gccgacaccg agacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440aagcgcctgg aagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500gccgccgggg cccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560gacaccctcg cctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggaaggccag 1620gaagtccagg aaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680aacgcccaca ggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740tccggcgccg ccatctgcga gatcaaggac tga 1773 29 1584 DNA Streptomycescoelicolor 29 atgaccgttt tggatgaggc gccgggcgag ccgacggacg cgcgcgggcgggtggccgag 60 ctgcacggga tccgtgcagc ggcgctcgcc gggccgagtg agaaggcgacggcggcgcag 120 cacgccaagg gcaagctgac ggcacgtgag cgcatcgagc tgctcctggaccccggctcc 180 ttccgcgagg tcgagcagct gcgccggcac cgggcgaccg ggttcggcctggaggccaag 240 aagccgtaca ccgacggtgt catcaccggc tggggcacgg tcgagggccgcacggtcttc 300 gtctacgccc acgacttccg gatcttcggc ggcgcgctgg gcgaggcccacgccacgaag 360 atccacaaga tcatggacat ggccatcgcg gccggtgccc cgctggtgtcgctgaacgac 420 ggcgccggcg cccgtatcca ggagggcgtc agcgcgctcg ccgggtacggcggcatcttc 480 cagcgcaaca ccaaggcgtc cggcgtcatc ccgcagatca gcgtgatgctcggcccctgc 540 gcgggcggcg cggcctacag ccccgccctc accgacttcg tcttcatggtccgcgacacc 600 tcgcagatgt tcatcacggg cccggacgtc gtcaaggcgg tcaccggcgaggagatcacg 660 cagaacggtc tgggcggcgc cgacgtgcac gccgagacgt ccggcgtgtgccacttcgcc 720 tacgacgacg aggagacctg cctcgccgag gtccgctacc tcctctccctcctcccgcag 780 aacaaccggg agaacccgcc ccgcgccgag tcctccgacc ccgtggaccgccgctcggac 840 accctcctcg acctggtccc ggcggacggc aaccgcccgt acgacatgaccaaggtcatc 900 gaggaactcg tcgacgaggg cgagtacctg gaggtccacg agcgttgggcccgcaacatc 960 atctgcgcgc tggcccgtct cgacgggcgg gtcgtgggca tcgtcgccaaccagccgcag 1020 gccctggccg gtgtcctgga catcgaggcg tcggagaagg cggcccgcttcgtccagatg 1080 tgcgacgcct tcaacatccc gatcatcact cttctggacg tacccggcttcctgcccggc 1140 gtcgaccagg agcacggcgg gatcatccgc cacggcgcca agctgctctacgcgtactgc 1200 aacgcgaccg tgccccggat ctcgctgatc ctgcgcaagg cgtacggaggtgcttacatc 1260 gtcatggaca gccagtccat cggcgccgac ctcacctacg cctggccgaccaacgagatc 1320 gccgtcatgg gcgcggaagg tgccgcgaac gtcatcttcc gccggcagatcgccgacgcc 1380 gaggaccccg aggccatgcg ggcgcgcatg gtcaaggagt acaagtccgagctgatgcac 1440 ccctactacg cggccgaacg cggtctggtc gacgacgtca tcgaccccgccgaaacccgc 1500 gaggtgctga tcacgtccct ggcgatgctc cacaccaagc acgccgacctgccctcccgc 1560 aagcacggca acccgccgca gtga 1584 30 198 DNA Streptomycescoelicolor 30 atgtcccctg ccgacatccg cgtcgagaag ggccacgccg agcccgaggaagtcgccgcc 60 atcacggccc tcctcctggc ccgcgccgcc gcccgccccg ccgagatcgcgccgacccac 120 ggcggcggcc gcgcccgcgc cggctggcgc cgcctggaac gcgagccgggcttccgcgcc 180 ccgcacagct ggcgctga 198 31 527 PRT Streptomycescoelicolor 31 Met Thr Val Leu Asp Glu Ala Pro Gly Glu Pro Thr Asp AlaArg Gly 1 5 10 15 Arg Val Ala Glu Leu His Gly Ile Arg Ala Ala Ala LeuAla Gly Pro 20 25 30 Ser Glu Lys Ala Thr Ala Ala Gln His Ala Lys Gly LysLeu Thr Ala 35 40 45 Arg Glu Arg Ile Glu Leu Leu Leu Asp Pro Gly Ser PheArg Glu Val 50 55 60 Glu Gln Leu Arg Arg His Arg Ala Thr Gly Phe Gly LeuGlu Ala Lys 65 70 75 80 Lys Pro Tyr Thr Asp Gly Val Ile Thr Gly Trp GlyThr Val Glu Gly 85 90 95 Arg Thr Val Phe Val Tyr Ala His Asp Phe Arg IlePhe Gly Gly Ala 100 105 110 Leu Gly Glu Ala His Ala Thr Lys Ile His LysIle Met Asp Met Ala 115 120 125 Ile Ala Ala Gly Ala Pro Leu Val Ser LeuAsn Asp Gly Ala Gly Ala 130 135 140 Arg Ile Gln Glu Gly Val Ser Ala LeuAla Gly Tyr Gly Gly Ile Phe 145 150 155 160 Gln Arg Asn Thr Lys Ala SerGly Val Ile Pro Gln Ile Ser Val Met 165 170 175 Leu Gly Pro Cys Ala GlyGly Ala Ala Tyr Ser Pro Ala Leu Thr Asp 180 185 190 Phe Val Phe Met ValArg Asp Thr Ser Gln Met Phe Ile Thr Gly Pro 195 200 205 Asp Val Val LysAla Val Thr Gly Glu Glu Ile Thr Gln Asn Gly Leu 210 215 220 Gly Gly AlaAsp Val His Ala Glu Thr Ser Gly Val Cys His Phe Ala 225 230 235 240 TyrAsp Asp Glu Glu Thr Cys Leu Ala Glu Val Arg Tyr Leu Leu Ser 245 250 255Leu Leu Pro Gln Asn Asn Arg Glu Asn Pro Pro Arg Ala Glu Ser Ser 260 265270 Asp Pro Val Asp Arg Arg Ser Asp Thr Leu Leu Asp Leu Val Pro Ala 275280 285 Asp Gly Asn Arg Pro Tyr Asp Met Thr Lys Val Ile Glu Glu Leu Val290 295 300 Asp Glu Gly Glu Tyr Leu Glu Val His Glu Arg Trp Ala Arg AsnIle 305 310 315 320 Ile Cys Ala Leu Ala Arg Leu Asp Gly Arg Val Val GlyIle Val Ala 325 330 335 Asn Gln Pro Gln Ala Leu Ala Gly Val Leu Asp IleGlu Ala Ser Glu 340 345 350 Lys Ala Ala Arg Phe Val Gln Met Cys Asp AlaPhe Asn Ile Pro Ile 355 360 365 Ile Thr Leu Leu Asp Val Pro Gly Phe LeuPro Gly Val Asp Gln Glu 370 375 380 His Gly Gly Ile Ile Arg His Gly AlaLys Leu Leu Tyr Ala Tyr Cys 385 390 395 400 Asn Ala Thr Val Pro Arg IleSer Leu Ile Leu Arg Lys Ala Tyr Gly 405 410 415 Gly Ala Tyr Ile Val MetAsp Ser Gln Ser Ile Gly Ala Asp Leu Thr 420 425 430 Tyr Ala Trp Pro ThrAsn Glu Ile Ala Val Met Gly Ala Glu Gly Ala 435 440 445 Ala Asn Val IlePhe Arg Arg Gln Ile Ala Asp Ala Glu Asp Pro Glu 450 455 460 Ala Met ArgAla Arg Met Val Lys Glu Tyr Lys Ser Glu Leu Met His 465 470 475 480 ProTyr Tyr Ala Ala Glu Arg Gly Leu Val Asp Asp Val Ile Asp Pro 485 490 495Ala Glu Thr Arg Glu Val Leu Ile Thr Ser Leu Ala Met Leu His Thr 500 505510 Lys His Ala Asp Leu Pro Ser Arg Lys His Gly Asn Pro Pro Gln 515 520525 32 65 PRT Streptomyces coelicolor 32 Met Ser Pro Ala Asp Ile Arg ValGlu Lys Gly His Ala Glu Pro Glu 1 5 10 15 Glu Val Ala Ala Ile Thr AlaLeu Leu Leu Ala Arg Ala Ala Ala Arg 20 25 30 Pro Ala Glu Ile Ala Pro ThrHis Gly Gly Gly Arg Ala Arg Ala Gly 35 40 45 Trp Arg Arg Leu Glu Arg GluPro Gly Phe Arg Ala Pro His Ser Trp 50 55 60 Arg 65

1. A nucleic acid comprising a nucleic acid sequence which encodes AccBpolypeptide, or a nucleic acid sequence complementary thereto, whereinsaid AccB polypeptide has the amino acid sequence set out in FIG. 12Aand/or the amino acid sequence encoded by the nucleic acid sequence setout in FIG. 12B.
 2. The nucleic acid of claim 1, wherein said nucleicacid sequence is as set out in FIG. 12B, or is complementary thereto. 3.The nucleic acid of claim 1, further comprising a nucleic acid sequencewhich encodes AccE polypeptide, or a nucleic acid sequence complementarythereto, wherein said AccE polypeptide has the amino acid sequence setout in FIG. 13A and/or the amino acid sequence encoded by the nucleicacid sequence set out in FIG. 13B.
 4. The nucleic acid of claim 3,wherein said nucleic acid sequence is as set out in FIG. 13B, or iscomplementary thereto.
 5. The nucleic acid of claim 1, furthercomprising a nucleic acid sequence which encodes AccA1 polypeptide, or anucleic acid sequence complementary thereto, wherein said AccA1polypeptide has the amino acid sequence set out in FIG. 11A and/or theamino acid sequence encoded by the nucleic acid sequence set out in FIG.11B.
 6. The nucleic acid of claim 5, wherein said nucleic acid sequenceis as set out in FIG. 11B, or is complementary thereto.
 7. The nucleicacid of claim 1, further comprising a nucleic acid sequence whichencodes AccA2 polypeptide, or a nucleic acid sequence complementarythereto, wherein said AccA2 polypeptide has the amino acid sequence setout in FIG. 11A and/or the amino acid sequence encoded by the nucleicacid sequence set out in FIG. 11B.
 8. The nucleic acid of claim 7,wherein said nucleic acid sequence is as set out in FIG. 11B, or iscomplementary thereto.
 9. The nucleic acid of claim 1 wherein saidnucleic acid sequence which encodes AccB polypeptide is in operativeassociation with a regulatory sequence for constitutive or inducibleexpression of said AccB polypeptide in Streptomyces species.
 10. Thenucleic acid of claim 9 wherein said regulatory sequence comprises thetipA inducible promoter.
 11. The nucleic acid of claim 3 wherein saidnucleic acid sequence which encodes AccE polypeptide is in operativeassociation with a regulatory sequence for constitutive or inducibleexpression of said AccE polypeptide in Streptomyces species.
 12. Thenucleic acid of claim 11 wherein said regulatory sequence comprises thetipA inducible promoter.
 13. The nucleic acid of claim 5 wherein saidnucleic acid sequence which encodes AccA1 polypeptide is in operativeassociation with a regulatory sequence for constitutive or inducibleexpression of said AccA1 polypeptide in Streptomyces species.
 14. Thenucleic acid of claim 13 wherein said regulatory sequence comprises thetipA inducible promoter.
 15. The nucleic acid of claim 7 wherein saidnucleic acid sequence which encodes AccA2 polypeptide is in operativeassociation with a regulatory sequence for constitutive or inducibleexpression of said AccA2 polypeptide in Streptomyces species.
 16. Thenucleic acid of claim 15 wherein said regulatory sequence comprises thetipA inducible promoter.
 17. A vector comprising the nucleic acidsequence set out in FIG. 12B under the control of the tipA promoter,whereby said vector is capable, after incorporation into Streptomycescoelicolor, of causing expression of AccB polypeptide having the aminoacid sequence set out in FIG. 12A and/or the amino acid sequence encodedby the nucleic acid sequence set out in FIG. 12B.
 18. The vector ofclaim 17, further comprising the nucleic acid sequence set out in FIG.13B under the control of said tipA promoter, whereby said vector iscapable, after incorporation into Streptomyces coelicolor, of causingexpression of AccE polypeptide having the amino acid sequence set out inFIG. 13A and/or the amino acid sequence encoded by the nucleic acidsequence set out in FIG. 13B.
 19. A Streptomyces coelicolor straincomprising the vector of claim
 17. 20. A Streptomyces coelicolor straincomprising the vector of claim
 18. 21. A method of producing apolyketide, the method comprising: providing a polyketide-producingstrain of Streptomyces coelicolor into which the vector of claim 17 hasbeen introduced; culturing said strain under conditions suitable forpolyketide synthesis; and extracting said polyketide from the cellculture medium.
 22. The method of claim 21, further comprising the stepof purifying said polyketide.
 23. The method of claim 22, furthercomprising the step of formulating said polyketide as a pharmaceutical.24. The method of claim 21 wherein said polyketide is an antibiotic. 25.A method of producing a polyketide, the method comprising: providing apolyketide-producing strain of Streptomyces coelicolor into which thevector of claim 18 has been introduced; culturing said strain underconditions suitable for polyketide synthesis; and extracting saidpolyketide from the cell culture medium.
 26. The method of claim 25,further comprising the step of purifying said polyketide.
 27. The methodof claim 26, further comprising the step of formulating said polyketideas a pharmaceutical.
 28. The method of claim 27, wherein said polyketideis an antibiotic.
 29. A method of modifying a polyketide-producingstrain of a Streptomyces species to increase production of saidpolyketide, the method comprising modifying said strain to express, orto increase expression of, nucleic acid according to claim
 1. 30. Themethod of claim 29, wherein said polyketide is an antibiotic.
 31. Themethod of claim 29, wherein said Streptomyces species is selected fromthe group consisting of S. coelicolor, S. violaceoruber, S. lividans andS. parvulus.
 32. The method of claim 31, wherein said strain is selectedfrom the group consisting of ATCC 12434, ATCC 19832, S. coelicolor A3(2)and S. lividans
 66. 33. The method of claim 31, wherein saidStreptomyces species is S. coelicolor.
 34. The method of claim 32,wherein said strain is S. coelicolor A3 (2).
 35. The method of claim 29,further comprising modifying said strain to express, or to increaseexpression of, nucleic acid according to claim
 3. 36. The method ofclaim 29, further comprising modifying said strain to express, or toincrease expression of, nucleic acid according to claim
 5. 37. Themethod of claim 29, further comprising modifying said strain to express,or to increase expression of, nucleic acid according to claim
 7. 38. Amodified strain of a Streptomyces species, produced according to themethod of claim
 29. 39. A method of producing a polyketide, the methodcomprising: providing the modified Streptomyces strain of claim 38;culturing said strain under conditions suitable for polyketidesynthesis; and extracting said polyketide from the cell culture medium.40. The method of claim 39, further comprising the step of purifyingsaid polyketide.
 41. The method of claim 40, further comprising the stepof formulating said polyketide as a pharmaceutical.
 42. A method ofincreasing acetyl-CoA carboxylase (ACCase) activity in a strain ofStreptomyces coelicolor, the method comprising modifying said strain toexpress, or to increase expression of, nucleic acid encoding AccBpolypeptide, said nucleic acid having the nucleic acid sequence set outin FIG. 12B.
 43. The method of claim 42, further comprising modifyingsaid strain to express, or to increase expression of, nucleic acidencoding AccE polypeptide, said nucleic acid having the nucleic acidsequence set out in FIG. 13B.
 44. The method of claim 42, furthercomprising modifying said strain to express, or to increase expressionof, nucleic acid encoding AccA1 polypeptide, said nucleic acid havingthe nucleic acid sequence set out in FIG. 11B.
 45. The method of claim42, further comprising modifying said strain to express, or to increaseexpression of, nucleic acid encoding AccA2 polypeptide, said nucleicacid having the nucleic acid sequence set out in FIG. 11B.
 46. Themethod of claim 42, wherein the strain is S. coelicolor A3(2).
 47. Amethod of increasing production of a polyketide in Streptomycescoelicolor, the method comprising culturing said cells in the presenceof exogenous malonate.
 48. The method of claim 47, wherein the malonateis present at a concentration of at least about 0.1%.
 49. The method ofclaim 48, wherein the malonate is present at a concentration of at leastabout 0.5%.
 50. The method of claim 49, wherein the malonate is presentat a concentration of at least about 1%.
 51. The method of claim 47wherein the polyketide is an antibiotic.